Researchers have examined the effects of mineral pitch (MP), an Ayurvedic medicine that is applied to body ailments and is commonly found in the Himalayan mountain region. The treatment proved successful in preventing cancer in a multi-faceted manner, by inducing apoptosis (programmed cell death) and inhibiting cell proliferation.
A study published in Biomed Central attempts to verify the anti-cancer properties of natural products. Hepatocellular Carcinoma (HCC), a form of neoplasm and the most common type of liver cancer, is considered one of the leading causes of death among cancer-related medical issues. Chemotherapies used to treat HCC have only proved to be successful in a limited number of patients and treatment also faces a concern of cancer cells developing resistance. Chemotherapeutic drugs are composed of 50% natural products, causing researchers to gain heavy interest in testing these products for their ability to prevent cancer. Of these natural products, mineral pitch was assessed by this study. The dark brown-coloured humic matter resides in the Himalayan Mountains in the vicinity of India and Nepal. MP is known to cure an array of ailments from wounds to bone fractures. The mineral contains fulvic acid and humic acid which account for its activity. The claim that mineral pitch possesses anti-cancer effects is tested by this study.
MP was collected from the village Matela located in far-western Nepal. Once brought back to the laboratory, a stock solution was made by dissolving 2 mg/ml of MP in distilled water. Hepatic cancer cells were cultured and supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The cells were then incubated with varying concentrations of MP for 24 hours, and cancer cell growth was observed.
Mineral Pitch was found to affect reactive oxygen species (ROS) and nitrogen oxide (NO) levels as well. Increased levels of ROS place oxidative stress on the cells causing molecular damage leading to apoptosis (programmed cell death). Increased levels of NO lead to the opening of pores in the mitochondria, allowing calcium to be released and reduces the mitochondrial membrane potential; this chain reaction also results in apoptosis of cells. To test for ROS and NO levels in the cancer cells, cells were pre-incubated with one of N-acetyl cysteine (NAC) or Nitroarginine methyl ester (L-NAME) for one hour prior to the addition of MP. After staining with EDTA, cells were counted followed by the calculation of apoptosis induction to determine how many cells were undergoing programmed cell death.
The level of ROS production was measured using cultured and incubated cells with the addition of 2’, 7’-dihydrochloroflurorescein acetate (DCFH-DA) solution. The fluorescence of DCFH-DA labelled cells was examined using flow cytometry analysis. NO production was measured from the cells. The media was then collected and used for the estimation of the production of NO using a Nitrate/Nitrite Colorimetric Assay Kit. RNA isolation, cDNA synthesis, and polymerase chain reaction (PCR) were carried out and the relative expression was calculated.
Cell viability was determined by looking for the presence of c-myc (a protein required for cell transcription and survival). Colony formation was tracked using crystal violet dissolved in ethanol staining. Mathematical calculations from the TUNEL assay were used to determine the percentage of cells undergoing apoptosis. Proteins were then isolated and a Western blot analysis was performed to determine the levels of c-myc.
The effect of MP on cell proliferation and survival was tested. When the cancer cells were incubated with MP, proliferation significantly decreased and became directly proportional to the increasing concentration of MP. There was a maximum 73.3% reduction in cell proliferation when 1000 μg/ml of MP was added. There was an observed inhibition in cell number as well as colony formation. These values suggest that MP holds strong anti-proliferative properties and can therefore be helpful in preventing cancer.
The effect of MP on apoptosis was also tested. TUNEL assay was performed to detect DNA fragmentation which results in apoptosis. Apoptosis induction was found to increase with increasing concentrations of MP. Cells incubated with an MP concentration of 1000 μg/ml were found to contain 80.1% apoptotic cells. The treatment was confirmed by Annexin V and PI exclusion assays. Flow cytometry results confirmed the TUNEL assay findings.
HCC is known to be highly drug resistant. This resistance develops due to enhanced DNA repair capacity. MP tests were all conducted using hepatic cancer cells and the positive results suggest that the natural product may be one of the few effective treatments for this type of cancer. These positive results can be attributed to MP’s ability to induce ROS generation and NO production. Incubated cells with varying concentrations of MP were tested for their ROS and NO levels. There was a 49.8% increase in ROS production at 500 μg/ml of MP. This increase in ROS may be evidence for oxidative stress-induced apoptosis. The Griess reagent was used to test NO production through nitrite levels. 65.33% NO production was found when 1000 μg/ml MP was added to the incubated cells. As mentioned above, increased levels of ROS and NO induce programmed cell death leading to prevention of cancer by arresting uncontrolled cell division.
This research offers an insight into the potential use of natural remedies. With such positive results towards a major medical concern, further research and analysis can be implemented to confirm the effects of mineral pitch on preventing cancer. This may allow development of unique cancer therapies that would avoid the issue of resistance and the need for transplants. With funding for such research, more options of cancer treatment may become available and offer patients the opportunity to overcome the disease effectively.
Written By: Shrishti Ahuja, BSc