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Mineral pitch induces apoptosis and inhibits proliferation via modulating reactive oxygen species in hepatic cancer cells
© The Author(s). 2016
- Received: 7 January 2016
- Accepted: 18 May 2016
- Published: 27 May 2016
Mineral Pitch (MP) is a dark brown coloured humic matter originating from high altitude rocks. It is an Ayurvedic medicinal food, commonly used by the people of the Himalayan regions of Nepal and India for various body ailments.
The Huh-7 cells were treated with different concentrations of MP for 24 h, and both apoptosis and proliferation was determined by the TUNEL and MTT assays respectively. The formation of ROS and nitric oxide was analysed by DCFH-DA and Griess reagent respectively. The expression of miRNA-21 and miRNA-22 were checked by the real time PCR. Effect of miRNA-22 on proliferation and c-myc was studied by over-expressing miRNA-22 premiRs in Huh-7 cells.
We found that MP enhanced anti-cancer effects by inducing apoptosis and inhibiting proliferation. MP induced both ROS and NO, upon neutralizing them, there was a partial recovery of apoptosis and proliferation. MP also induced miRNA-22 expression, while miRNA-21 expression was inhibited. Over-expression of miRNA-22 resulted in a significant inhibition of proliferation. miRNA-22 directly targeted c-myc gene, thereby inhibited proliferation. These results clearly show that MP induces its anti-cancer activity by more than one pathway.
The data clearly indicate that MP induced apoptosis via the production of ROS, and inhibited proliferation by inducing miRNA-22 and inhibiting miRNA-21 in Huh-7 cells.
Hepatocellular Carcinoma (HCC) is a complex form of neoplasm, associated with many risk factors such as, Hepatitis B and C virus infection, non-alcoholic fatty liver disease (NAFLD), alcohol abuse, aflatoxins, diabetes, obesity, and genetic factors . HCC is the third leading cause of death among the cancer-related problems, and the prime cause of mortality among the cirrhosis patients .
Surgical resection is the best therapeutic preference for non-malignant primary liver tumour. The number of patients who undergo liver transplants are very less, which is due to the long waiting list, less number of transplant surgeons, donor availability problems and high cost. Several chemotherapeutic drugs have been assayed for the treatment of HCC . However, success has been achieved only in few patients, or some with inequitable response. The major scientific challenge against HCC is the limited response to the available chemotherapy and the development of resistance during the treatment. Resistance to chemotherapy may be due to an improved DNA repair capacity and greatly activated antioxidant enzymes in the cancer cells. HCC is considered highly resistant to the therapeutic agents, leading to the DNA damage .
Approximately, 50 % of the modern anticancer drugs used in the cancer chemotherapy have been originated from the natural products. Hence, the use of natural products in the development of new drugs has been a great interest for researchers.
Recently several work has been done with Traditional Chinese Medicine (TCM) for the treatment of cancer. TCM has been used as adjuvant therapy to inhibit cancer, when Western medicines cannot provide any treatment options. TCM is used in conjunction with chemotherapy and radiotherapy for inhibiting the toxic effects of the treatments, as well as improving overall efficacy [3, 4].
Mineral Pitch (MP), also called as shilajit in local vernacular, is a dark brown coloured humic matter that drips out of the high altitude rocks (above 1000 m) during summer months. It is believed that it can cure almost all body ailments [5–7]. MP is a natural medicinal food, mainly used to treat people with weakness, inflammation, bone fracture, bleeding and for wound healing . Since there is an insufficient number of medical facilities, eighty percent of total population mostly rely on the natural products for their primary health care needs .
MP is a humic matter, shown to contain fulvic acid and humic acid, which are responsible for its biochemical activities. In some previous studies humic matter has been reported to be anticancer agent as it inhibited the cancer cell growth and induced the apoptosis . Moreover the cytotoxic properties of humic acid was accompanied the ROS production  and NO synthesis . MP has been reported to be useful in reducing inflammation, arthritis, rheumatism, pain, ulcer, anxiety, stress, and diabetes . Recently, MP also has been reported as an antiviral agent against Herpes Simplex Virus (HSV)  however, many of its other potential functions including its effects on cancer, has not been studied so far.
In this study, the anticancer property of MP was determined in hepatic cancer cells. It is an essential ethno-medicinal food, has large demand due to many therapeutic benefits in remedial recipes. Since a large population of Nepal and India customarily consume MP, the present investigation might have some implication in understanding its therapeutic significance in HCC management.
Collection of Mineral Pitch (MP)
MP was collected from the village Matela, Baitadi district of far-western Nepal, from the local inhabitants. This drug was transported to the laboratory in a clean and sterile bag. The stock solution was made by dissolving 2 mg/ml in double distilled water and was stored after filtering with 0.44 μm membrane filter and used for all the experiments.
Cell culture and MP treatment
Huh-7 cells (1 × 106) were cultured in 6-well plates using Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Carlsbad, USA) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin. After 24 h, the cells were cultured in serum-free media and further incubated with different concentrations (0, 10, 20, 50, 100, 500, and 1000 μg/ml) of MP for 24 h. After incubation, the cells were washed and collected either for various assays, RNA isolation for RT-PCR or Western blots.
ROS and NO neutralization experiments
Huh-7 cells were cultured in 6-well plates and the medium was replaced with serum-free media and the cells were incubated with MP (0, 50 and 100 μg/ml) for a period of 24 h. For ROS and NO neutralizing experiments, once the cells were replenished with serum-free media, the cells were pre-incuated with either N-acetyl cysteine (NAC, 5 mM) or L-NG-Nitroarginine methyl ester (L-NAME, 10 μM) for 1 h followed by the addition of MP (100 μg/ml) for a further period of 24 h. After the incubation, the cells were washed with PBS and were dissociated using PBS-2 mM EDTA and then stained with Annexin V or PI. Immediately the positive cells were counted using flow cytometry and the apoptosis induction was calculated.
Cell viability assay
Colony formation assay
The 2.0 × 103 cells were seeded in 60 mm cell culture-treated dishes, along with the various concentrations of the MP (10–1000 μg/ml). The cells were incubated for 6 days, with a change of media and MP at every 24 h. At the end of 6 days, 0.5 % crystal violet dissolved in ethanol was used to stain the cells and the data were calculated .
Huh-7 cells were cultured (5 × 105 cells/well) for 24 h and the media was replaced with serum-free media along with different concentrations of MP for a further period of 24 h. A solution of 2’,7’-dihydrochloroflurorescein acetate (DCFH-DA) (10 μM) was added to the cells and incubated for 60 min in dark. After staining, the cells were collected using PBS containing 2 mM EDTA. The fluorescence of DCFH-DA labelled cells were examined using flow cytometry analysis.
The cells (5 × 105 cells per well) were incubated with different concentration of MP for 24 h. After the incubation, the media was collected and used for the estimation of the production of NO using Nitrate/Nitrite Colorimetric Assay Kit (Cayman, USA) as per the manufacturer’s instructions. The percent of nitrite production was calculated against untreated control.
Lipid peroxidation assay
After the MP treatment, the cells (5 × 105 cells per well) were lysed. The homogenate (1 ml) was mixed with 0.15 M Tris–Cl buffer (pH 7.4), 10 % trichloroacetic (TCA) and 50 mM thiobarbituric acid (TBA). This mixture was heated for 30 min at 80 °C, cooled and centrifuged for 10 min at 3000 rpm. The absorbance of the supernatant was measured against the blank (distilled water) at 530 nm in a UV spectrophotometer and MDA formed in the cells was determined.
Assay for cellular antioxidant enzymes
The cells were incubated with different concentration of MP. After the incubation, reduced glutathione (GSH), catalase and SOD activity assays were performed according to the methods described previously [16, 17]. The total cellular protein content was estimated using BCA reagent (Thermo Scientific, USA) as per the manufacturer’s instructions. The protein content was used for the calculation of the enzyme activities.
RNA isolation, cDNA synthesis, and Real-time PCR
Total RNA enriched with miRNA was extracted from Huh-7 cells using the mirVana miRNA isolation kit (Life Technologies, Carlsbad, CA, USA), following the manufacturer’s instructions. Reverse transcription was performed using the Universal cDNA synthesis kit (Exiqon, Vedbaek, Denmark). RT-PCR was done with SYBR Green and PCR master mix (Life Technologies). Cyber-green method was used for the real-time PCR using specific primers for both miRNA-21 and miRNA-22, and 5S RNA was used as an internal control as described previously [18, 19]. Each PCR was performed in duplicates and the data were normalized with endogenous 5S RNA levels. Relative expression were calculated using 2-ΔΔct values. All the experiments were repeated at least three times in triplicates.
MiRNA-22 transfection experiments
Huh-7 cells cells (3.5 × 105 cells/well) were plated in 6-well plates and transfected with either miRNA-22 (10nM) or a non-specific miRNA (10nM; a miRNA which does not inhibit any known mRNA) (Sigma Aldrich, USA) using siPORT miRNA transfection reagent (Invitrogen, USA) as per the manufacturer’s instructions. In parallel, the cells were transfected with fluorescein conjugated siRNA to check the transfection efficiency. After 24 h, the media was changed and allowed to grow for 48 h and then the cells were collected either for RNA isolation or protein isolation.
Protein isolation and Western blot analysis
After 72 h of transfection, the total cellular protein was isolated from the transfected cells using mammalian protein extraction buffer (Thermo Scientific, USA). Protein concentration was estimated using bicinconinic acid (BCA) protein estimation kit (Thermo Scientific). Samples (60 μg/lane) were run on a 12 % SDS-PAGE gels, and transferred to polyvinylidene fluoride membranes. Western blots were analyzed using antibodies for c-myc protein and β-actin (Cell signalling, USA). After washing and developing using ECL kit (Thermo Scientific), the protein bands were visualized.
All the experiments were conducted in duplicates and at least three independent experiments. The data were calculated and expressed as mean ± standard deviation (SD). The analysis of variances (ANOVA) was calculated among the groups followed by the Student’s t-test for the differences between the groups. The level of significance was computed and the values were considered significant when p < 0.05.
Results and discussion
MP contains 60–80 % humic matter (humic acid, fulvic acid and humin), and rest of the content may be secondary metabolites from the plant and/or animal origin . The presence of several contents might be due to the plant/animal origin of MP or addition of various medicinal plants (i.e. Ashwagandha, Triphala or Tulsi) during the cooking process, which could augment the therapeutic properties of MP against various health complaints. In the present study, we demonstrated the anticancer properties of MP, by performing proliferation and apoptosis assays.
MP inhibits cell proliferation
MP induces apoptosis
Resistance to chemotherapy in tumour cells is due to enhanced DNA repair capacity. HCC is considered to be a tumour which is highly resistant to agents attacking DNA. Several herbal composite formulas and natural components (Curcumin, Resveratrol, and Silibinin) have been shown to be beneficial for the cancer chemoprevention via inducing the DNA damage . In the present study, for the first time we have reported the anti-proliferative and pro-apoptotic properties of the MP in hepatic cancer cells.
MP induces both ROS generation and NO production
The Griess reagent is used to measure the level of NO produced in the cells. Since NO is highly unstable and gets converted to nitrite, the level of the nitrite was measured with increasing concentrations of the MP. The percentage of the NO production was 65.33, 61.2, 60.27, 51.26, 36 and 24.7 for 1000, 500, 200, 100, 50, and 20 μg/ml, reported respectively and these values were statistically significant (p < 0.01) (Fig. 3b). NO may lead to the cell death via inducing pro-apoptotic signals. During the lack of respiration, mitochondrial membrane potential is reduced, cytochrome c is released, the transition pores are opened and calcium is released by the increased NO level which eventually leads to apoptosis .
Effect of MP on cellular antioxidants
Superoxide dismutase (SOD) converts superoxide into H2O2 and O2. The catalase (CAT) enzyme catalyses the decomposition of H2O2 to H2O and O2 . These enzymes ensure that the cells effectively deal with ROS and free radicals, induced oxidative stress. We demonstrated that although MP simultaneously down-regulated SOD, CAT and while GSH was up-regulated in cancer cells, the cell death was increased. The falling level of the cellular antioxidants may be a reason for the augmented level of ROS in the cells.
The lipid peroxidation of the cancer cells was found to be increased in concentration dependent manner. There was a 2-fold increase with 50 and 100 μg/ml concentration of MP and there was a 3.3 fold increase in MDA levels with 500 and 1000 μg/ml concentration of MP respectively (Fig. 4d). It is the oxidative degradation of lipids by free radicals and ROS which capture electrons from the lipids of cell membranes, leading to the cell damage and finally results in the apoptosis . In this study, we have clearly shown the in-vitro anti-cancer properties of MP through generation of the ROS and NO, which was accompanied by the lipid peroxidation-induced apoptosis in hepatic cancer cells.
MP-induced ROS plays a role in proliferation and apoptosis
MP decreases the expression of miRNA-21 and increases the expression of miRNA-22
miRNA-21 is a specific oncomiR (cancer specific MiRNA), found more abundant in human cancers like lung, pancreas, skin, liver, gastric, cervical, thyroid, and various lymphatic and hematopoietic cancers. Inhibition of miRNA-21 in HCC lines reduced the phenotypic behaviours of cancer cells (e.g. decreased cell proliferation, migration and invasion). It contributes to the enhanced aggressiveness of HCC and consequently results in poor diagnosis in HCC patients. It induces the proliferation of cancer cells by the repressing the PTEN (a tumour suppressor gene) . Previously we have shown that over-expression of miRAN-21 increases the proliferation in heaptic cancer cells . The data suggest that MP might inhibit proliferation, at least in part, via inhibiting miRNA-21 in these cells.
miRNA-22 has been observed as a tumour suppressing agent in many cancers including HCC . A known target of miRNA-22 is histone deacetylase 4, which is known to have many important functions in cancer development and proliferation . MP treatment lead to the reduction of the miRNA-21 while it increased the level of the miRNA-22 expression significantly, which indicates the anti proliferative activities of MP via regulating the epigenetic factors.
Over-expression of miRNA-22 decreases proliferation in Huh-7 cells
Throughout the centuries of herbal and natural medicinal practice, there are a number of candidate drugs derived from the herbs or herbal compounds for chemotherapeutic approach against HCC. Certain herbal compounds have been found as an anti-HCC agent and are successfully in use for cancer therapy. In this manuscript we have shown that MP could be developed as a potential natural chemotherapeutic agent.
CAT, Catalase; DCFDA, 2’,7’ dichloro-fluorescein diacetate; DMSO, Dimethyl sulfoxide; L-NAME, L-NG-Nitroarginine methyl ester; MDA, malondialdehyde; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAC, N-acetyl cysteine; NO, Nitric oxide; PBS, Phosphate buffer saline; ROS, Reactive oxygen species; RT-PCR, Real time PCR; SOD, Superoxide dismutase; TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labelling.
We would like to thank the local ethnic people of far western Nepal for providing us the mineral pitch. We also express our gratitude to Mr. Nitin Sharma and Mr. Milind Dongardive for providing technical help.
The study was partly supported by extramural grants from Department of Biotechnology, Government of India, India and partly from the intramural funds provided by South Asian University, New Delhi, India.
Availability of data and materials
All the data that are pertinent to the manuscript have been included in the main text and in figures. No additional or supplementary data is available.
KP and SKV designed the study and drafted the manuscript. KP collected the mineral pitch from Nepal. KP also performed some of the cell culture and most of the Western blots. Most of the cell culture experiments and other experiments are performed by PG, AKY, AG and AA. PD has done all the miRNA-22 over-expression experiments. KP has written the manuscript and SKV interpreted the data as well as corrected the manuscript. All authors have read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Yes, we do give full consent for publication.
Ethics approval and consent to paticipate
This study only had used the cell lines for all the experiments. Hence no animals or humans were used. No ethics approval is needed and not relevant.
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