#MMP-3 ELISA

[bostorbio]

An enzyme proenzyme is released from the cells in the body that is known as MMP-3 (MMP-3 ELISA). The proenzyme has been shown to promote plasminogen activation, as previously reported.

[eannattoblog]

Lung Cancer Clinical Trials and Treatment- EAnnatto DeltaGold

Many conservative treatments of cancer like chemotherapy, surgery, and radiation have failed to eliminate lung cancer completely. In a report by World Health Organisation (WHO), dated 12 September 2018, it was observed that approximately1.8 million deaths happened due to Lung cancer that year which is the largest number as compared to other cancers! Henceforth it won’t be wrong to say that lung cancer is the deadliest cancer. Moreover lung cancer has been observed to be the 2nd most common cancer in both men and women and about 13% of all new cancers are lung cancers and trust me, a share of 13% amongst all cancers is a huge share. Lung cancer is the leading cause of cancer death among both men and women. Each year, more people die of lung cancer than of colon, breast, and prostate combined. Overall the chance that man will develop lung cancer in his lifetime is about 1 in 15 while for a woman; the risk is 1 in 17. These numbers include both smokers and non – smokers.

In the pursuit to fight cancer, researchers have discovered Tocotrienol which is supposed to demonstrate anti-cancer activities. A number of studies have been conducted over Annatto based Tocotrienol (DeltaGold – Eannatto). Several studies have been conducted on Tocotrienol for its effects against lung cancer like ‘Delta-Tocotrienol inhibits non-small lung cancer cell invasion via the inhibition of NF-kb, uPA activator, and MMP-9’, where it was observed that Delta-Tocotrienol attenuated tumor invasion and metastasis by the repression of MMP-9/uPA via downregulation of Notch-1 and NF-kB pathways and upregulation of miR-451, another study, ‘Tocotrienol – rich mixture inhibits cell proliferation and induces apoptosis vie down – regulation of the Notch – 1/NF – kB pathway in NSCLC cells’ in which the anticancer property, “inhibition of cell proliferation” of Tocotrienols was observed.

Study 1 - Delta-Tocotrienol hinder non-small lung cancer cell attack via the inhibition of NF-kb, uPA activator, and MMP-9

Non – small cell lung cancer (NSCLC) accounts for 85% of all lung cancer cell and can be classified into 3 sub – type’s squamous cell carcinoma, large cell carcinoma, and adrenocarcinoma. The initial stage of NSCLC has a 5 year survival rate of 55% but this rate reduces to less than 4% for cases diagnosed with remote metastasis. Matrix metalloproteinase 9 (MMP – 9) is an enzyme with 21 subtypes in humans which regulates cell migration, angiogenesis, adhesion, aggregation, and immune response in cancer. In this process, MMP – 9 is mainly accountable for humiliating collagen type 4 and elastin in basal membranes, facilitating lung cancer metastasis. High levels of MMP – 9 have also been reported in the serum of lung carcinoma patients. Thus, the modulation of MMP – 9 protein expressions and their activities would be excellent therapeutic targets for the inhibition of assault and metastasis processes in NSCLC.

Urokinase – type plasminogen activator (uPA), a serine proteinase, binds to the urokinase – type plasminogen activator receptor (uPAR) and transforms inactive plasmin and other protease, including MMP – 9, into their active forms.  Regulating uPA is one of the major approaches that can directly modulate MMP – 9 activities in cancer. Increased expression of the uPA system has been reported in NSCLC tissue as compared to common lung tissue. It has been observed that nuclear factor – kB (NF – kB) a transcription factor (TF) involved in cancer instigation and progression directly binds with the uPA promoter in vitro. The same study showed that the inhibition of Nf – kB activities decreased cell attack and uPA synthesis in NSCLC cells.

Previous studies showed that Delta – Tocotrienol (Eannatto – DetaGold) subdued NF – kB signaling pathways via the down regulation of Notch – 1, thereby decreasing the proliferation and metastatic/invasion potential, while inducing apoptosis of NSCLC cells in a concentration and time – dependent manner. This study investigated the anti - metastatic effect of Delta – Tocotrienol (Eannatto – DeltaGold) on NSCLC cells with the hypothesis that MMP – 9 – dependent invasion and metastasis of NSCLC cells were inhibited by the suppression of Notch – 1 – mediated NF – kB and uPA pathways and induction of miR – 451.

Results

1. It was showed that a concentration – dependent reduction in cell proliferation in cancer cells with the addition of Delta – Tocotrienol (Eannatto – DeltaGold).

2. Inhibition of cell invasion and migration by Delta – Tocotrienol (Eannatto – DeltaGold) was observed.

3. Inhibition of cell aggregation at 30 μM of Delta – Tocotrienol (Eannatto – DeltaGold) in cancer cells and adhesion capabilities by DeltaGold were demonstrated.

4. DeltaGold was observed to suppress expression of MMP – 9.

5.  Delta – Tocotrienol (Eannatto – DeltaGold) was observed to increase the expression of miR – 451. miR – 451 plays a key role on different aspects of carcinogenesis in lung cancer including tumor growth, invasion, radio resistance, and chemoresistance.

6.Delta – Tocotrienol (Eannatto – DeltaGold) was also observed to inhibit expression uPA and Notch – 1 pathway proteins.

7. Inhibition of NF – kB DNA – binding activity was demonstrated by Delta – Tocotrienol (Eannatto – DeltaGold).

 Study 2 –Tocotrienol – rich blend reduce cell proliferation and persuade apoptosis via down – regulation of the Notch – 1/NF – kB pathway in NSCLC cells

It has been observed that aberrant Notch – 1 expression have been reported in lung cancer patients and could potentially be a beneficial molecular/therapeutic target against NSCLC. Tocotrienols, isomers of Vitamin E, have been shown to exhibit antitumor activity via inhibition of different signaling pathways in tumor cells. This study was conducted on commercially available Tocotrienols (a mixture of isomers) on the Noth – 1 pathway in NSCLC, adenocarcinoma (A549) and squamous cell lung cancer (H520) cell lines. A dose – dependent decline in all growth, cell migration, and tumor invasiveness was observed in both cancer cell lines with the addition of Tocotrienols. A significant induction of apoptosis was also observed using Annexin V stain in flow cytometry analysis.  Since Tocotrienols significantly affected proliferation, apoptosis, migration, and invasiveness, reverse transcription polymerase chain reaction and Western blot analysis were used to explore the molecular mechanisms responsible for the regulations by testing the expression of Notch – 1 and its downstream genes. A dose – dependent decrease in expression of proteins was observed in Notch – 1, Hes – 1, Survivin, and Bcl – XL. In addition, a mechanism was founded linking the NF – kB pathway and Notch – 1 down regulation from Tocotrienols inhibits cell growth, migration, and tumor cell invasiveness via down regulation of Notch – 1 and NF – kB while inducing apoptosis. Hence, these commercially available Tocotrienol – rich mixture could potentially be an effective supplementation for lung cancer prevention.

Two different NSCLC cell lines, representing squamous cell carcinoma (H520) adenocarcinoma (A549) were cultured in Rosewell Park Memorial Institute (RPMI), medium (Mediatech, Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin in 5% CO2 and 37 degree Celsius. Tocotrienol – rich capsules provided by Carotino (Kula Lumpur, Malaysia), containing 21.3% Tocopherols and 78.7% Tocotrienols, were used in this study. The Tocotrienols in the capsules contained 26.7% Alpha – Tocotrienol, 3.3% Beta – Tocotrienol,, 38.1% Gamma – Tocotrienol and 10.6 Delta – Tocotrienol isomers, whereas the remainder 21,3% composed of Alpha – Tocopherol isomer. The media containing dimethyl sulfoxide (DMSO) (vehicle control) or different concentrations of TRMC diluted from a 100 mg/mL stock solution were used as experimental treatment media for cell culture. The final concentrations of treatment media is expressed as the amount of TRMC (mg) in 1 mL of RPMI media (mg/mL).

Results

1.  Anti – proliferative effects of TRMC on NSCLC cell lines were analyzed using MTS assay where anti – proliferative  effects of the Tocotrienol mixture were demonstrated.

2.  Cell death detection histone/deoxyribonucleic acid (DNA) enzyme linked immunosorbent assay (ELISA) Kit from Roche (Palo Alto, CA, USA) was used to detect apoptosis in NSCLC cells. A549 and H520 cells were seeded into 6 – well plates at the density of 1×105 and 1×106 cells, respectively and after an overnight incubation, cells were treated with control medium or treatment medium 9different concentrations of TRMC)for 72 hour. Further proceedings showed that apoptosis or programmed cell death was induced in the cancer cells when acted upon by the mixture of Tocotrienols.

3. Tocotrienols were observed to induce apoptosis in lung cancer in cell lines.

4.  Inhibition of cell assault and migration by Tocotrienols was observed. The effect of TRMC on tumor cell invasion and migration was evaluated using Matrigel invasion and wound – healing assays. TRMC concentrations (0.4 - 0.12 mg/mL) resulted in a significantly decreased saturation of lung cancer cells through the Matrigel – coated membrane as compared to the control cells, confirming that TRMC compact the invasion capacity of lung cancer cells. For further confirmation of anti – migratory effects of TRMC, the wound – healing assay was performed. The results of the wound – healing assay exposed that there was decline in cell migration from custom – made wounds with 0.8 mg/mL of TRMC after 30 hours of incubation. In contrast, there was a significant wound healing in the control cells without TRMC, under the same incubation conditions.

5. Down regulation of the Notch – 1 and its target gene expressions by Tocotrienols was observed.

6. Inhibition of NF – kB DNA binding activity with Tocotrienols was observed.

Summary

Why Tocotrienol?

·  Antioxidants, especially Tocotrienol was observed to demonstrate anti-cancer activity against lung cancer cells.

·  Angiogenesis which is the process of formation of blood vessels in cancer cells like in your brain cancer. Tocotrienol promotes cancer cell death to a very great extent and good results of anti-angiogenesis property of Tocotrienols have been observed against lung cancer cells in the study.

· Apoptosis or programmed cell death is the process of elimination and death of cancer cells. All isoforms of Tocotrienols have been observed to induce apoptosis in lung cancer cells through the activation of caspase-8 and mitochondrial cyt.c release. Tocotrienol was also reported to tempt apoptosis in human lung adenocarcinoma that harbors Ras mutation.

·Cell Proliferation is the process by which cancer cells copy their DNA and divide into two cancer cells during mitosis and rapidly multiply into more cancer cells. It was observed that Delta-Tocotrienol reticent lung cancer cell proliferation, induce cancer cell death and prevented cell cancer invasion.

· Chemoprevention and anti-cancer activity against lung cancer have been observed in Tocotrienols. Delta-Tocotrienol was observed to exert anti-cancer activity in lung cancer by elevating microRNA, miR-34a, that led to the downregulation of Notch-1 and its downstream targets such as Bcl-2, cyclin D1, and survivin. Moreover, it was also observed that a redox-silent analogue of Alpha-Tocotrienol, 6-O-carboxypropyl-alpha-tocotrienol was found to possess higher anti-cancer potential than Tocotrienol in A549 lung cancer cells.

· Hypoxia adaptation of lung cancer cells was observed to be suppressed by Tocotrienol through the inhibition of Src-induced Akt activation and decreased HIF-2 alpha.

· Cancer stem cell death has been observed by the action of Tocotrienols especially Delta – Tocotrienols (DeltaGold – Eannatto). Even after chemotherapies, radiation and surgeries, there are stem cells of those cancerous tissues left revolving in your body which can lead to your cancer coming back. Henceforth, their death is very necessary and Tocotrienols have been observed to kill cancer stem cells.

 Dosage

·  Under the study, 900 mg/day of Tocotrienols were used to treat lung cancer cells and no adverse effects were observed and the death of breast cancer cells was witnessed.

· Substances that complement Tocotrienol for cancer include Vitamins C, D, Selenium, B complex.

Why Tocotrienol and Not Tocopherol?

·Tocopherol, the enemy of Tocotrienol: Tocopherol has been observed to ease lung cancer inhibition, inhibits absorption, reduces adipose storage, and compromises cholesterol and triglyceride reduction. Tocopherol hinders the functioning of Tocotrienol and even when they are consumed simultaneously, Tocopherol obstructs all the functions of Tocotrienol.

·Tocopherol, the antagonist in liver cancer treatment:It has been observed that Tocopherol not only interfered with the functioning of Tocotrienol but also showed unsafe effects during the treatment.

· Tocotrienol, the protector of State: Tocotrienol has more mobility than Tocopherol due to its small structure so it can cover a larger area targeting more number of lung cancer cells.

· Small structure and less molecular weight: The higher anti-oxidant activity of Tocotrienols is due to their small structure and less molecular weight which help in their integration of the cell, unlike Tocopherols.

References

·  Tocotrienols: Latest Cancer Research in Vitamin E by Barrie Tan, Ph.D., and Anne M.Trias, MS.

·  Tocotrienols: The Promising Analogues of Vitamin Efor Cancer Therapeutics

https://doi.org/10.1016/j.phrs.2018.02.017

· Delta-tocotrienol inhibits non-small-cell lung cancer cell invasion via the inhibition of NF-κB, uPA activator, and MMP-9.

https://www.ncbi.nlm.nih.gov/pubmed/30100736

· Tocotrienol – rich mixture inhibits cell proliferation and induces apoptosis via down – regulation of the Notch – 1/NF – kB pathway in NSCLC cells.

https://www.dovepress.com/tocotrienol-rich-mixture-inhibits-cell-proliferation-and-induces-apopt-peer-reviewed-fulltext-article-NDS

Note:

1. To read studies in detail, follow the references and links given.

2. The dosages given must not be taken as the advice of a medical practitioner. Consult your physician for the optimum dosage to be consumed.

[phgq]

Hinundayan clings on its reputation as Covid-free town

#PHinfo: Hinundayan clings on its reputation as Covid-free town

HINUNDAYAN, Southern Leyte, Mar. 8 (PIA) -- The local government unit here has continued to hold on to its title as a Covid-free municipality, with only three confirmed cases thus far, the least number in the entire province.

But that was a fallback status, as the LGU almost went on record as the only place in the province with zero COVID-19 patient.

“We really wanted to maintain our image as the municipality without a single case -- until the very first case came out on December 9, 2020,” Hinundayan Mayor Elisa Cadingan shared with local reporters during the field Kapihan sa PIA session last week.

Her sharing carried a sense of intimacy, with hints of an opportunity lost in her pensive looks, yet she emphasized that the LGU met the challenge squarely.

Still, she was grateful to note that so far, they managed to keep the numbers low, as low as three (3), the lowest province-wide, and those three individuals had since gone home to their families, re-integrated into the mainstream of the community.

On the issue of acquiring the Covid vaccines, Mayor Cadingan said she was looking for a budget of about P7 million to P8 million as their 70 percent counterpart to the 30 percent budget prepared by the provincial government for the municipality.

When told that the national government through the Department of Health (DOH) will be the one to give 50 percent of the vaccines for their priority population -- the other 50 percent to be shared 70-30 by the LGU and province -- Cadingan declined to comment, inasmuch as the DOH has yet to deliver the items.  (ldl/mmp/PIA8-Southern Leyte)

***

References:

* Philippine Information Agency. "Hinundayan clings on its reputation as Covid-free town." Philippine Information Agency. https://pia.gov.ph/news/articles/1068836 (accessed March 08, 2021 at 10:25PM UTC+08).

* Philippine Infornation Agency. "Hinundayan clings on its reputation as Covid-free town." Archive Today. https://archive.ph/?run=1&url=https://pia.gov.ph/news/articles/1068836 (archived).

[healthtimetaylor]

These findings offer curcumin as a new therapeutic agent with the potential of regulating astrocyte-mediated inflammatory diseases in the CNS.

PMID:  Int Immunopharmacol. 2014 Sep ;22(1):230-5. Epub 2014 Jul 3. PMID: 24998635 Abstract Title:  Study of curcumin immunomodulatory effects on reactive astrocyte cell function. Abstract:  Multiple sclerosis (MS) is considered an inflammatory and neurodegenerative disease of the central nervous system (CNS) which most often presents as relapsing-remitting episodes. Recent evidence suggests that activated astrocytes play a dual functional role in CNS inflammatory disorders such as MS. In this study, we tried to induce anti-inflammatory functions of astrocytes by curcumin. The effects of curcumin were examined on human a astrocyte cell line (U373-MG) induced by lipopolysaccharide (LPS) in vitro. Matrix metalloproteinase (MMP)-9 activity was assessed by gelatin zymography. Cytokine levels were evaluated by quantitative ELISA method and mRNA expression was measured by real-time PCR. We found that curcumin decreased the release of IL-6 and reduced MMP-9 enzyme activity. It down-regulated MCP-1 mRNA expression too. However, curcumin did not have significant effects on the expression of neurotrophin (NT)-3 and insulin-like growth factor (IGF)-1 mRNAs. Results suggest that curcumin might beneficially affect astrocyte population in CNS neuroinflammatory environment lean to anti-inflammatory response and help to components in respects of CNS repair. Our findings offer curcumin as a new therapeutic agent with the potential of regulating astrocyte-mediated inflammatory diseases in the CNS.

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[taliemsite-blog]

Intra-amniotic infection (IAI) is a major cause of preterm labor and adverse neonatal outcome. We evaluated amniotic fluid (AF) proteolytic cascade forming biomarkers in relation to microbial invasion of the amniotic cavity (MIAC) and IAI in preterm pregnancies with intact membranes. Material and Methods. Amniocentesis was made to 73 women with singleton pregnancies; 27 with suspected IAI; and 46 controls. AF biomarkers were divided into three cascades: Cascade 1: matrix metalloproteinase-8 (MMP-8), MMP-9, myeloperoxidase (MPO), and interleukin-6; Cascade 2: neutrophil elastase (HNE), elafn, and MMP-9; Cascade 3: MMP-2, tissue inhibitor of matrix etalloproteinases-1 (TIMP-1), MMP-8/TIMP-1 molar ratio, and Creactive protein(CRP). MMP-8 was measured by an immunoenzymometric assay and the others were measured by ELISA. Standard biochemical methods, molecular microbiology, and culture techniques were used. Results. MMP-8, MMP-9, MPO, elafn, and TIMP-1 concentrations were higher in IAI suspected cases compared to controls and also in IAI suspected cases with MIAC compared to those without MIAC when adjusted by gestational age at amniocentesis.

#biology

[cancersfakianakis1]

Non-invasive profiling of protease-specific elastin turnover in lung cancer: biomarker potential

Abstract

Purpose

Elastin is a signature protein of lungs. Increased elastin turnover driven by altered proteolytic activity is an important part of lung tumorigenesis. Elastin-derived fragments have been shown to be pro-tumorigenic, however, little is known regarding the biomarker potential of such elastin fragments. Here, we present an elastin turnover profile by non-invasively quantifying five specific elastin degradation fragments generated by different proteases.

Methods

Elastin fragments were assessed in serum from patients with stage I–IV non-small cell lung cancer (NSCLC) (n = 40) and healthy controls (n = 30) using competitive ELISAs targeting different protease-generated fragments of elastin: ELM12 (generated by matrix metalloproteinase MMP-9 and -12), ELM7 (MMP-7), EL-NE (neutrophil elastase), EL-CG (cathepsin G) and ELP-3 (proteinase 3).

Results

ELM12, ELM7, EL-NE and EL-CG were all significantly elevated in NSCLC patients (n = 40) when compared to healthy controls (n = 30) (ELM12, p = 0.0191; ELM7, p < 0.0001; EL-NE, p < 0.0001; EL-CG, p < 0.0001). ELP-3 showed no significant difference between patients and controls (p = 0.8735). All fragments correlated positively (Spearman, r: 0.69–0.81) when compared pairwise, except ELM12 (Spearman, r: 0.042–0.097). In general, all fragments were detectable across all stages of the disease.

Conclusions

Elastin fragments generated by different proteases are elevated in lung cancer patients compared to healthy controls but differ in their presence. This demonstrates non-invasive biomarker potential of elastin fragments in serum from lung cancer patients and suggests that different pathological mechanisms may be responsible for the elastin turnover, warranting further validation in clinical trials.

http://bit.ly/2Dpguvh

[battybat-boss]

Carnosine had chondroprotective effects on type 2 diabetes mellitus-induced osteoarthritis.

PMID:  Front Pharmacol. 2018 ;9:598. Epub 2018 Jun 6. PMID: 29928231 Abstract Title:  Carnosine Prevents Type 2 Diabetes-Induced Osteoarthritis Through the ROS/NF-κB Pathway. Abstract:  The anti-inflammatory and antioxidant capacity of carnosine (CAR) has been investigated in autoimmune diseases. The aim of this study was to evaluate the potential protective effects of oral CAR supplements to ameliorate type 2 diabetes mellitus (T2DM)-induced osteoarthritis (OA) in rats and its mechanism.Seventy male Sprague-Dawley rats were randomly divided into the control group (CG,= 10) and the T2DM group (= 60). A rat model of T2DM was established using a high fat diet and streptozotocin (30 mg/kg, i.p.). The 41 rats that developed T2DM were chosen and randomly divided into four groups: T2DM-induced OA group (OAG,= 11), and the T2DM-induced OA with low, moderate, and high-doses of CAR for 8 weeks group (CAR-L, CAR-M, and CAR-H,= 10). After 13 weeks, all rats were evaluated by enzyme-linked immunosorbent assay (ELISA), histology, immunohistochemistry, and western blotting. Fibroblast-like synoviocytes (FLSs) were obtained from the knee joints of all rats. The effects of CAR on the inflammatory response in interleukin (IL)-1β-stimulated FLSs under a high glucose environment were evaluated by real-time quantitative polymerase chain reaction, western blotting, flow cytometry, and immunofluorescence.The results of ELISA (IL-1β and tumor necrosis factor-α), the histological evaluation (Mankin and OARSI score), western blotting [COL2A1, matrix metalloproteinase (MMP)-3, MMP-13, IL-1β, and nuclear factor-kappaB (NF-κB) p65], and immunohistochemistry (COL2A1, MMP-3, and MMP-13) indicated that oral CAR attenuated the development of T2DM-induced OA and suppressed the inflammatory response. Moreover, CAR alleviated MMP-3 and MMP-13 expression levels by decreasing reactive oxygen species content and suppressing nuclear translocation of NF-κB p65 on IL-1β-induced FLSs in a high glucose environment.These findings indicate that oral CAR had chondroprotective effects on T2DM-induced OA through the reactive oxygen species (ROS)/NF-κB pathway.

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[lopezdorothy70-blog]

Carnosine had chondroprotective effects on type 2 diabetes mellitus-induced osteoarthritis.

PMID:  Front Pharmacol. 2018 ;9:598. Epub 2018 Jun 6. PMID: 29928231 Abstract Title:  Carnosine Prevents Type 2 Diabetes-Induced Osteoarthritis Through the ROS/NF-κB Pathway. Abstract:  The anti-inflammatory and antioxidant capacity of carnosine (CAR) has been investigated in autoimmune diseases. The aim of this study was to evaluate the potential protective effects of oral CAR supplements to ameliorate type 2 diabetes mellitus (T2DM)-induced osteoarthritis (OA) in rats and its mechanism.Seventy male Sprague-Dawley rats were randomly divided into the control group (CG,= 10) and the T2DM group (= 60). A rat model of T2DM was established using a high fat diet and streptozotocin (30 mg/kg, i.p.). The 41 rats that developed T2DM were chosen and randomly divided into four groups: T2DM-induced OA group (OAG,= 11), and the T2DM-induced OA with low, moderate, and high-doses of CAR for 8 weeks group (CAR-L, CAR-M, and CAR-H,= 10). After 13 weeks, all rats were evaluated by enzyme-linked immunosorbent assay (ELISA), histology, immunohistochemistry, and western blotting. Fibroblast-like synoviocytes (FLSs) were obtained from the knee joints of all rats. The effects of CAR on the inflammatory response in interleukin (IL)-1β-stimulated FLSs under a high glucose environment were evaluated by real-time quantitative polymerase chain reaction, western blotting, flow cytometry, and immunofluorescence.The results of ELISA (IL-1β and tumor necrosis factor-α), the histological evaluation (Mankin and OARSI score), western blotting [COL2A1, matrix metalloproteinase (MMP)-3, MMP-13, IL-1β, and nuclear factor-kappaB (NF-κB) p65], and immunohistochemistry (COL2A1, MMP-3, and MMP-13) indicated that oral CAR attenuated the development of T2DM-induced OA and suppressed the inflammatory response. Moreover, CAR alleviated MMP-3 and MMP-13 expression levels by decreasing reactive oxygen species content and suppressing nuclear translocation of NF-κB p65 on IL-1β-induced FLSs in a high glucose environment.These findings indicate that oral CAR had chondroprotective effects on T2DM-induced OA through the reactive oxygen species (ROS)/NF-κB pathway.

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[homeopathylove-blog]

Anticancer potential of Conium Maculatum Extract Against Cancer Cells in Vitro: Drug-DNA Interaction and its Ability to Induce Apoptosis through ROS Generation

AbstractObjective:

Conium maculatum extract is used as a traditional medicine for cervix carcinoma including homeopathy. However, no systematic work has so far been carried out to test its anti-cancer potential against cervix cancer cells in vitro. Thus, in this study, we investigated whether ethanolic extract of conium is capable of inducing cytotoxicity in different normal and cancer cell lines including an elaborate study in HeLa cells.

Materials and Methods:

Conium’s effects on cell cycle, reactive oxygen species (ROS) accumulation, mitochondrial membrane potential (MMP) and apoptosis, if any, were analyzed through flow cytometry. Whether Conium could damage DNA and induce morphological changes were also determined microscopically. Expression of different proteins related to cell death and survival was critically studied by western blotting and ELISA methods. If Conium could interact directly with DNA was also determined by circular dichroism (CD) spectroscopy.

Results:

Conium treatment reduced cell viability and colony formation at 48 h and inhibited cell proliferation, arresting cell cycle at sub-G stage. Conium treatment lead to increased generation of reactive oxygen species (ROS) at 24 h, increase in MMP depolarization, morphological changes and DNA damage in HeLa cells along with externalization of phosphatidyl serine at 48 hours. While cytochrome c release and caspase-3 activation led HeLa cells toward apoptosis, down-regulation of Akt and NFkB inhibited cellular proliferation, indicating the signaling pathway to be mediated via the mitochondria-mediated caspase-3-dependent pathway. CD-spectroscopy revealed that Conium interacted with DNA molecule.

Conclusion:

Overall results validate anti-cancer potential of Conium and provide support for its use in traditional systems of medicine.

Keywords: Apoptosis, Conium maculatum, drug-DNA interaction, proliferation, reactive oxygen species

INTRODUCTION

Conium maculatum is an extremely poisonous flowering weed, known as Hemlock and belongs to the family Apiaceae. Conium contains several pyridine alkaloids like coniine, N-methylconiine, conhydrine, pseudoconhydrine and gamma-coniceine, precursors of some other hemlock alkaloids.[1] The structures of these alkaloids are shown in Figure 1a. Among these, the most notable one is coniine, the properties of which are similar to nicotine. It disrupts the functions of the central nervous system by binding with nicotinic acetylcholine receptors.[2,3] Though this plant is highly toxic in nature, its extract had been used as a traditional remedy for different diseases since a long time.[4] As for example, Conium is the main remedy for prostate gland and swelling of the testis. In homeopathy, it is used as a remedy for breast cancer and cancer of cervix uteri,[4] but its action has not yet been scientifically validated except for the report that it can inflict DNA damage by generating reactive oxygen species (ROS).[5] In this study, we contemplate to elucidate the probable mechanism of action of the drug in inducing apoptosis in the cervix cancer cell line HeLa.

[healthtimetaylor]

Licochalcone D induces apoptosis and inhibits migration and invasion in human melanoma A375 cells.

PMID:  Oncol Rep. 2018 May ;39(5):2160-2170. Epub 2018 Mar 20. PMID: 29565458 Abstract Title:  Licochalcone D induces apoptosis and inhibits migration and invasion in human melanoma A375 cells. Abstract:  The aim of the present study was to determine the effects of Licochalcone D (LD) on the apoptosis and migration and invasion in human melanoma A375 cells. Cell proliferation was determined by sulforhodamine B assay. Apoptosis was assessed by Hoechst 33258 and Annexin V‑FITC/PI staining and JC‑1 assay. Total intracellular reactive oxygen species (ROS) was examinedby DCFH‑DA. Wound healing and Transwell assays were used to detect migration and invasion of the cells. The activities of matrix metalloproteinase (MMP‑2 and MMP‑9) were assessed via gelatin zymography. Tumor growth in vivo was evaluated in C57BL/6 mice. RT‑PCR, qPCR, ELISA and western blot analysis were utilized to measure the mRNA and protein levels. Our results showed that LD inhibited the proliferation of A375 and SK‑MEL‑5 cells in a concentration‑dependent manner. After treatment with LD, A375 cells displayed obvious apoptotic characteristics, and the number of apoptotic cells was significantly increased. Pro‑apoptotic protein Bax, caspase‑9 and caspase‑3 were upregulated, while anti‑apoptotic protein Bcl‑2 was downregulated in the LD‑treated cells. Meanwhile, LD induced the loss of mitochondrial membrane potential (ΔΨm) and increased the level of ROS.ROS production was inhibited by the co‑treatment of LD and free radical scavenger N‑acetyl‑cysteine (NAC). Furthermore, LD also blocked A375 cell migration and invasion in vitro which was associated with the downregulation of MMP‑9 and MMP‑2. Finally, intragastric administration of LD suppressed tumor growth in the mouse xenograft model of murine melanoma B16F0 cells. These results suggest that LD may be a potential drug for human melanoma treatment by inhibiting proliferation, inducing apoptosis via the mitochondrial pathway and blocking cell migration and invasion.

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[healthtimetaylor]

Suppressive effects of the standardized extract of Phyllanthus amarus on type II collagen-induced rheumatoid arthritis.

PMID:  Curr Pharm Biotechnol. 2018 ;19(14):1156-1169. PMID: 30539691 Abstract Title:  Suppressive Effects of the Standardized Extract of Phyllanthus amarus on Type II Collagen-induced Rheumatoid Arthritis in Sprague Dawley Rats. Abstract:  BACKGROUND: Standardized extract of Phyllanthus amarus has been shown to possess inhibitory effects on cellular and humoral immune responses in Wistar-Kyoto rats and Balb/c mice.OBJECTIVE: In the present study, the standardized extract of P. amarus was investigated for its suppressive effects on type II collagen-induced rheumatoid arthritis (TCIA) in Sprague Dawley rats.METHOD: The major components of the extracts, lignans and phenolic compounds were analysed by using a validated reversed phase HPLC and LC-MS/MS. A rheumatoid arthritis rat model was induced by administering a bovine type II collagen emulsion subcutaneously at the base of tail, on day 0 and 7 of the experiment. Effects of the extract on severity assessment, changes in the hind paw volume, bone mineral density, body weight and body temperature were measured. Concentrations of cytokines (TNF-α, IL-1β, IL-1α, IL-6) released, matrix metalloproteinases (MMP-1, MMP-3 MMP-9) and their inhibitor (TIMP-1), haematological and biochemical changes were also measured. ELISA was used to measure the cytokines and proteinases in the rat serum and synovial fluid according to manufacturer's instructions.RESULTS: The extract dose-dependently modulated the progression in physical parameters (i.e. decrease in body weight, increase in body temperature, reduced hind paw volume, reduced the severity of arthritis), bone mineral density, haematological and biochemical perturbations, serum cytokines production and levels of matrix metalloproteinases and their inhibitor in the synovial fluid. Histopathological examination of the knee joint also revealed that the extract effectively reduced synovitis, pannus formation, bone resorption and cartilage destruction.CONCLUSION: The results suggest that the oral administration of a standardized extract of P. amarus was able to suppress the humoral and cellular immune responses to type II collagen, resulting in the reduction of the development of TCIA in the rats.

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[healthtimetaylor]

A hydroalcoholic extract of oak acorn shell acts as a potent antiangiogenic agent.

PMID:  Pharm Biol. 2013 Mar ;51(3):361-8. Epub 2012 Nov 8. PMID: 23137183 Abstract Title:  In vitro inhibition of angiogenesis by hydroalcoholic extract of oak (Quercus infectoria) acorn shell via suppressing VEGF, MMP-2, and MMP-9 secretion. Abstract:  CONTEXT: Angiogenesis is an essential factor for cancer progression. Although more attention is paid in angiogenesis on its role in cancer biology, many other non-neoplastic diseases are also angiogenic-dependent. Recently, there is motivation to control cancer via inhibition of angiogenesis.OBJECTIVE: Quercus infectoria Olivier var (Fagaceae) (oak) is a plant whose different parts, such as its fruit shell, have been used extensively as a traditional drug in the west part of Iran. Although some biological properties of oak are determined, its effects on angiogenesis are unclear. So, we investigated the antiangiogenic effects of oak acorn shell.MATERIALS AND METHODS: Fresh oak acorns were collected, and after authentication; hydroalcoholic extract of acorn shells (5, 10, 20, 30, 40, 60, 80, and 100μg/ml) was used for evaluation of its cytotoxicity, antiproliferative, and antiangiogenic effects in vitro. Also, effects of the extract on vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2) and MMP-9 secretion were assayed using enzyme-linked immunosorbent assay (ELISA)and gelatin zymography.RESULTS: Treatment with hydroalcoholic extract in eight doses resulted in a significant decrease of endothelial cell proliferation and angiogenesis with an IC₅₀ value of ~20 μg/ml, without any toxic effect. At 40 μg/ml, the extract inhibited MMP-9 activity; however, a dose-dependent reduction (60-80 µg/ml) in MMP-2 activity was seen. VEGF secretion was decreased with increase in the concentration of the extract from 5 to 100 μg/ml.DISCUSSION AND CONCLUSION: This study indicated that hydroalcoholic extract of oak acorn shell acts as a potent antiangiogenic agent which exerts its inhibitory effect mainly through downregulation of essential mediators such as VEGF and MMPs.

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The protective effects of thymoquinone on lung damage caused by cigarette smoke.

PMID:  Biotech Histochem. 2019 Nov 5:1-8. Epub 2019 Nov 5. PMID: 31687851 Abstract Title:  The protective effects of thymoquinone on lung damage caused by cigarette smoke. Abstract:  Chronic obstructive pulmonary disease (COPD) is characterized by systemic inflammation that usually is caused by exposure to noxious particles or gases. Thymoquinone (TQ) prevents the production of inflammatory mediators, such as thromboxane B2 and leukotriene, by altering arachidonic acid metabolism. We investigated the preventive and curative effects of TQ on lung damage in rats caused by cigarette smoke (CS). We used 50 adult male rats, 30 of which were exposed to CS every day for 3 months. TQ in dimethylsulfoxide (DMSO) was administered intraperitoneally (i.p.) every day to ten animals to investigate the protective effects of TQ, and to ten other animals during the last 21 days to investigate the curative effect. Ten rats received saline for the last 21 days. Ten subjects were untreated controls. Ten controls that were not exposed to CS received TQ for the last ten days. Serum IL-8, IL-6, IL-1β and MMP-9 levels were measured using ELISA. IL-1β and IL-8 levels were elevated in the group exposed to CS compared to controls. IL-8 levels were decreased in the group that received only TQ compared to controls, which indicated the anti-inflammatory effect of TQ. The apoptotic index (AI) was increased in all groups that were exposed to CS compared to controls. The AI index was decreased in the group that received TQ for the last 21 days compared to the other CS groups. AI was increased in the group that received TQ daily compared to the other CS groups. Our findings indicate that TQ exerts curative effects for the inflammation caused by CS and may prevent apoptosis if administered in appropriate doses; however, long term TQ or DMSO exposure may produce cumulative toxic effects.

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Paeonol may be a potential therapeutic agent in the treatment of osteoarthritis.

PMID:  Inflammation. 2017 Oct ;40(5):1698-1706. PMID: 28695367 Abstract Title:  Paeonol Inhibits IL-1β-Induced Inflammation via PI3K/Akt/NF-κB Pathways: In Vivo and Vitro Studies. Abstract:  Paeonol, the main active component isolated from the root of Paeonia suffruticosa, has been reported to have anti-inflammatory properties. However, the effects of paeonol on osteoarthritis (OA) remain unclear. The aim of this study was to investigate the anti-inflammatory effects and mechanism of paeonol in IL-1β-induced human OA chondrocytes as well as mice OA models. Human OA chondrocytes were pretreated with different concentrations of paeonol 2 h prior to IL-1β (10 ng/mL) stimulation for 24 h. Nitric oxide (NO) production was determined by Griess method. The levels of prostaglandin E2 (PGE2), matrix metalloproteinase 1 (MMP-1), MMP-3, and MMP-13 were assessed by ELISA. Inducible nitric oxide synthase (INOS), COX-2, and PI3K/Akt/NF-κB-related signaling molecules production were measured by Western blot. In vivo, mice OA models were established by destabilization of the medial meniscus. One month after surgery, mice in paeonol-treated group were given intraperitoneal injection of paeonol in 30 mg/kg every day, while mice of vehicle-treated group were injected with DMSO under the same conditions. Hematoxylin and eosin as well as Safranin-O staining were applied to assess the severity ofcartilage lesions. The results showed that pretreatment with paeonol could inhibit IL-1β-induced NO and PGE2 production. Meanwhile, the overproduction of INOS, COX-2, MMP-1, MMP-3, and MMP-13 were also reversed by paeonol. Moreover, paeonol was found to inhibit IL-1β-induced NF-κB activation, PI3K, and AKT phosphorylation. In vivo, treatment with paeonol exhibited less cartilage degradation and lower Osteoarthritis Research Society International scores in mice OA models. In conclusion, these results suggest that paeonol may be a potential therapeutic agent in the treatment of OA.

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Carnosol may represent a promising therapeutic candidate that canmodulate breast cancer growth and metastasis.

PMID:  Front Oncol. 2019 ;9:743. Epub 2019 Aug 8. PMID: 31456939 Abstract Title:  Carnosol, a Natural Polyphenol, Inhibits Migration, Metastasis, and Tumor Growth of Breast Cancer via a ROS-Dependent Proteasome Degradation of STAT3. Abstract:  We have previously demonstrated that carnosol, a naturally occurring diterpene, inhibitedcell viability and colony growth, as well as induced cell cycle arrest, autophagy and apoptosis in human triple negative breast cancer (TNBC) cells. In the present study, we evaluated the ability of carnosol to inhibit tumor growth and metastasis. We found that non-cytotoxic concentrations of carnosol inhibited the migration and invasion of MDA-MB-231 cells in wound healing and matrigel invasion assays. Furthermore, gelatin zymography, ELISA, and RT-PCR assays revealed that carnosol inhibited the activity and downregulation the expression of MMP-9. Mechanistically, we demonstrated that carnosol suppressed the activation of STAT3 signaling pathway through a ROS-dependent targeting of STAT3 to proteasome-degradation in breast cancer cells (MDA-MB-231, Hs578T, MCF-7, and T47D). We show that blockade of proteasome activity, by MG-132 and bortezomib, or ROS accumulation, by N-acetylcysteine (NAC), restored the level of STAT3 protein. In addition, using chick embryo tumor growth assay, we showed that carnosol significantly and markedly suppressed tumor growth and metastasis of breast cancer xenografts. To the best of our knowledge, this is the first report which shows that carnosol specifically targets signal transducer and activator of transcription 3 (STAT3) for proteasome degradation in breast cancer. Our study further provide evidence that carnosol may represent a promising therapeutic candidate that canmodulate breast cancer growth and metastasis.

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Red clover extract has a chondroprotective effect on inflammation and may be a potential preventative agent for osteoarthritis progression.

PMID:  Cells Tissues Organs. 2018 ;206(1-2):95-105. Epub 2019 Jan 31. PMID: 30703768 Abstract Title:  The Effect of the Prethanol Extract of Trifolium pratense Leaves on Interleukin-1β-Induced Cartilage Matrix Degradation in Primary Rat Chondrocytes. Abstract:  BACKGROUND: Osteoarthritis (OA) is a degenerative joint disease, characterized by cartilage degradation and inflammation. The proinflammatory cytokine, interleukin (IL)-1β, plays a crucial role in the pathogenesis of OA by inducing the release of other catabolic factors that contribute to cartilage degradation. Trifolium pratense L. (red clover) has been used as a medicinal plant in many countries and as a source of nutraceuticals to alleviate the symptoms of menopause. Ob-jectives: In this study, we aimed to evaluate the anticatabolic effect of 40% prethanol extract of T. pratense (40% PeTP) on IL-1β-stimulated chondrocytes.METHODS: Primary rat chondrocytes were pretreated with 40% PeTP for 1 h before stimulation with IL-1β (20 ng/mL). The production of nitrite, prostaglandin E2 (PGE2), and aggrecan was measured by using Griess reagent and ELISA. Protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, A disintegrin and metalloproteinase with thrombospondin motif (ADAMTS)-4, mitogen-activated protein kinase (MAPK), and the nuclear factor (NF)-κB p65 subunit was measured by using Western blotting.RESULTS: PeTP (40%) significantly inhibited the IL-1β-induced expression of nitrite, iNOS, PGE2, COX-2, MMP-1, MMP-3, MMP-13, and ADAMTS-4 in isolated primary rat chondrocytes. Furthermore, 40% PeTP decreased the IL-1β-induced degradation of aggrecan, the phosphorylation of MAPKs, and the nuclear translocation of the NF-κB p65 subunit.CONCLUSION: These results suggested that 40% PeTP has a chondroprotective effect on inflammation and may be a potential preventative agent for OA progression.

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