International Journal of Bio-Technology and Research (IJBTR) ISSN(P): 2249-6858; ISSN(E): 2249-796X Vol. 4, Issue 2, Apr 2014, 7- 14 © TJPRC Pvt. Ltd.
PIPERINE INDUCES DOWN REGULATION OF BCL2 AND UP REGULATION OF BAD IN SMOKELESS TOBACCO INDUCED HUMAN ORAL SQUAMOUS CELL CARCINOMA (SCC4)
NIKHIL KHURANA, RAJVARDHAN SINGH, ANJANA SINGH & VISHWAS TRIPATHI School of Biotechnology, Gautam Buddha University, Greater Noida, Uttar Pradesh, India
ABSTRACT
Tobacco consumption has been linked with the high incidence of head and neck cancer. Head and neck cancer is among one of the most common cancers worldwide. Piperine, a major alkaloid constituent of black pepper, has been previously reported to have anti-cancer activity in different cancer cell lines. It is a biosafe neutraceutical. The effect of piperine against smokeless tobacco induced head and neck cancer is not currently known. Therefore, in this study, we investigated the anti-cancer effect of piperine on smokeless tobacco induced head and neck cancer cells. We showed that piperine inhibited the proliferation of SCC4 head and neck cancer cells in a dose and time dependent manner. Furthermore, we observed the effect of piperine on apoptotic marker genes. Our study suggests that Piperine induces down regulation of BCL2 and up Regulation of BAD and Tp53 in smokeless tobacco induced human head and neck squamous cell carcinoma (SCC4) cells. Taken together, these results support further investigation of piperine as a potential therapeutic agent in the treatment of head and neck cancer.
KEYWORDS: Head and Neck Cancer, Piperine, Smokeless Tobacco, Squamous Cell Carcinoma (SCC4)
INTRODUCTION
Head and neck squamous cell carcinoma (HNSCC) ranks among the top 10 most common cancers worldwide. It includes cancers of the mouth, nose, sinuses, salivary glands, throat, and lymph nodes in the neck. Most begin in the moist tissues that line the mouth, nose and throat. Of the several causes investigated for cancer, the use of tobacco has shown strong and consistent associations with cancer at several sites of the body and tobacco use is the single largest cause of cancer globally [1, 2]. Tobacco is used by more than 1.3 billion people worldwide. As per the WHO estimates it kills more than 5 million people annually and this number is likely to increase 8 million by 2030 [3,4]. The primary reason for high incidence of head and neck cancer is the indiscriminate use of chewing tobacco in its various forms. Tobacco can be used in two forms smoked and smokeless. The tobacco smoked is used in cigarettes, cigars, or pipes and the smokeless tobacco is used in multiple forms namely gutkha, khaini, naswar. It has been classified as a carcinogen by International Agency of Research in Cancer (IARC) (Warnakulasuriya, Ralhan, 2007) (Cogliano, Straif , Baan, Grosse, Secretan, 2004). Smokeless tobacco (ST) consumption has been linked to the high incidence of HNSCC (Warnakulasuriya, Ralhan, 2007) (Sapkota, Berger, Vogel, 2010) (McClave , Whitney , Thorne , Mariolis , Dube , 2010).
The relation between smoking and HNSCC is extensively studied but the role of smokeless tobacco in HNSCC is yet to be precisely explored. The International Agency for Research on Cancer (IARC) has listed 28 carcinogens present in smokeless tobacco (9). This list includes tobacco-specific N-nitrosamines (TSNA), volatile N-nitrosamines, volatile aldehydes, polycyclic aromatic hydrocarbons (PAH), certain lactones, urethane, metals, polonium-210 and uranium-235
www.tjprc.org [email protected] 8 Nikhil Khurana, Rajvardhan Singh, Anjana Singh & Vishwas Tripathi and -238 (9) (Hecht, 1988) (Hecht, Hoffmann, 1988). Moreover, Smokeless tobacco contains nicotine and its use can lead to nicotine addiction [12]. Some smokeless products deliver higher doses of nicotine than cigarettes (Winstanley, 2009). Besides addiction, nicotine is also reported to inhibit apoptosis induced by anticancer drugs by regulating cell proliferation, angiogenesis (Xu, Huang, Pan, Zhang, Li, Zhang, 2007). It has been reported that Nicotine induces resistance to chemotherapy-induced apoptosis by modulating mitochondrial signaling in lung cancer cells (Zhang, Kamdar, Le, Rosen, Upadhya, 2009). Recently, it has been observed that khaini, a type of ST, induces PI3K/Akt NF-kB and STAT3 pathway in head and neck cancer cells (Macha, Matta ,Chauhan , Siu, Ralhan, 2011). Therefore, it is important to identify agents that can abrogate these effects of Smokeless tobacco in head and neck cancer cells for developing therapies to prevent and treat ST-related head and neck cancer. In recent decades, active components isolated from botanical sources have attracted great attention in the biomedical area because of their high effectiveness and low toxicity. One such compound is Piperine, which is a major alkaloid constituent of black pepper. It has previously been reported to have anti-cancer activity in variety of cancer cell lines. However, the effect of piperine against smokeless tobacco induced head and neck cancer is not yet known. Therefore, the objective of this study is to determine the anti-cancer activities of piperine, in smokeless tobacco induced head and neck cancer as well as to determine the underlying molecular mechanisms of its action.
MATERIAL AND METHODS Cell Culture
Human Head and neck cancer cell line (SCC4), derived from a squamous carcinoma of human tongue was borrowed from All India Institute of Medical Sciences, New Delhi, India (AIIMS). SCC4 is an established Head and Neck Cancerous cell line. SCC4 cells have a point mutation at codon 51 CCC to TCC (Sakai, Tsuchida, 1992) (Kim, Li, Bertolami, Cherrick, Park, 1993). SCC4 was grown in monolayer cultures in Dulbecco’s modified eagle medium (DMEM) (Himedia) supplemented with 10% Fetal bovine serum (FBS) (Himedia),1 mM L-glutamine, 1X sodium pyruvate, 1X vitamins, 1 mM minimum essential medium (MEM), 100 mg/ml streptomycin and 100 U /ml penicillin in a humidified incubator (5% carbon-dioxide, 95% air) at 37°C. The cells were first treated with different doses of smokeless tobacco followed by incubation with different concentrations of piperine (3.125, 6.25, 12.5, 25, 50 and 100μM) at 24h, 48h, and 96h.
Chemicals and Drugs
Piperine was purchased from Sigma-Aldrich Canada (Oakville, ON, Canada). A 100-mM stock solution of piperine was prepared in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and stored at −80°C. Phosphate-buffered saline (PBS), ethidium bromide (EtBr), agarosegel and 0.4% trypan blue dye from was obtained from Himedia Co.
Preparation of Smokeless Tobacco Extract (ST)
Khani (cured tobacco) was purchased from the local market. 25 g of tobacco was finely powdered and homogenized in 225 ml of distilled water and the mixture was stirred on a magnetic stirrer for two hours and then allowed to stand for 24 hours at 37˚C. Thereafter, supernatant was collected after centrifugation at 5000 g for 20 minutes. The extract was sterilized by passing it through a 0.22 mm filter and stored at 4˚C till use. The final concentration of tobacco in aqueous extract was estimated to be 2.7 g%.
Impact Factor (JCC): 2.8872 Index Copernicus Value (ICV): 3.0 Piperine Induces Down Regulation of BCL2 and up Regulation of Bad in 9 Smokeless Tobacco Induced Human Oral Squamous Cell Carcinoma (SCC4) Cytotoxicity Assay (MTT Assay)
The effect of piperine on the growth of SCC4 cells was examined using MTT assay. EZ count TM, MTT Cell Assay kit supplied by Himedia Co. was used for the determination of the effect of the compound and for calculating 3 IC50.Cells were sub cultured in 96-well plates at a density of 5×10 cells per well with and without piperine (3.125, 6.25, 12.5, 25, 50 and 100μM) for 24 h, 48 h, or 96 h in a final volume of 200 μl. Then, the medium was removed and 20 μl of MTT (5 mg/mL in PBS) was added to the fresh medium. After 2 h incubation at 37 °C, 100 μl of solublization buffer was added to each well and plates were agitated for 1 min. The formazan crystals were dissolved in 100 ml of solubalization buffer supplied with the kit. Spectrophotometric absorbance was measured at 570 nm. The percentage viability was calculated as the following formula: (viable cells) %=( OD of drug-treated sample/OD of untreated sample) ×100.
Total RNA Extraction and cDNA Synthesis
RNA was extracted by RNA easy lipid tissue kit supplied by Qiagen .c DNA synthesis was performed using Quantitect ® Reverse Transcription Kit supplied by Qiagen.
Agarose Gel Electrophoresis
The PCR products were resolved by electrophoresis in 1.5% Agarose gel in 0.5XTBE (Tris-Borate-EDTA) buffer. 100 bp ladders was used as molecular weight by UV transilluminator and photographed. Gel electrophoresis was carried out to confirm the primers specificity and amplification of PCR products marker. After staining the gel with ethidium bromide, fragments were visualized.
DETERMINATION OF CDNA EXPRESSION BY REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION (RT-PCR)
HotStarTaq Master Mix Kit supplied by Qiagen was employed to perform semi-quantitative PCR on the Qantras thermo-cycler, Germany, applying the following thermal-cycling conditions: 15 min at 95°C as first denaturation and Hot-start enzyme activation, followed by 40 cycles at 94°C for 1 min (denaturation), 60°C for 1 min (annealing), 72°C for 1 min (elongation) and final elongation at 72 °C for 10 min. The PCR amplification was performed in 20μl reaction containing 10μl HotStarTaq Master Mix, 1μl Forward primer (0.4μM), 1μl Reverse primer (0.4μM), 4μl first-strand cDNA (100ng) and 4 μl RNase free water. All band densities in the RT-PCR method were analyzed by the 1-D Multi Lane Densitometry program by using an Alpha Imager 2000 (Alpha Innotech Corp., San Leandro, CA, USA).
Table 1: Nucleotide Sequences of the Primers Used for Amplification of cDNA Target Gene Primer Sequence Product Length (bp) BCL2 5ꞌ-ATGTGTGTGGAGAGCGTCAA-3ꞌ 123 BAD 5ꞌ-CCCAGAGTTTGAGCCGAGTG-3ꞌ 249 TP53 5ꞌ-GTTCCGAGAGCTGAATGAG-3ꞌ 133
STATISTICAL ANALYSIS
Data are expressed as mean; standard deviation and graph preparation were done using Microsoft Office Excel 2007 software to estimate the reproducibility of the assay. The P-value of <0.05 was considered statistically significant for student's t-test analysis. Compared to the controls (untreated cells), the lower dose of piperine (3.125μM) (difference not
www.tjprc.org [email protected] 10 Nikhil Khurana, Rajvardhan Singh, Anjana Singh & Vishwas Tripathi
significant, p>0.05) and the higher dose of piperine (100μM) (p<0.001) decreased in total cell number (Figure 1, 2). IC50 of piperine after 24h, 48h and 96h was calculated.
RESULTS
SCC4 cells were grown in two sets, one treated with smokeless tobacco only and another treated with smokeless tobacco followed by treatment with piperine. The first set of cells which were treated with different concentrations of smokeless tobacco showed cell proliferation at conc. of 6.25µg/ml with maximum growth suggesting that tobacco promotes cell proliferation in SCC4 oral cancer cell lines (Figure 1). The second set of cells was first treated with smokeless tobacco followed by treatment with different dosage of piperine (3.125, 6.25, 12.5, 25, 50 and 100μM) for different time points (24hrs, 48hrs and 72 hrs) and MTT assay was performed. We found that piperine showed strong cytotoxic effect on smokeless tobacco treated SCC4 cell lines with 400µg/ml LC50 at 24hrs, 200µg/ml LC50 at 48hrs
100µg/ml LC50 at 96hrs suggesting that the cytotoxic effect of piperine increases with time (Figure 2). In order to further confirm the anti-proliferative effect of piperine in smokeless tobacco treated SCC4 cells, we observed the expression of certain pro-apoptotic and anti-apoptotic marker genes (BCL2, BAD and Tp53) by semi quantitative analysis. RNA was extracted from SCC4 cells treated with smokeless tobacco and smokeless tobacco followed by treatment with piperine. The results of MTT assay were in accordance with the results at RNA level. SCC4 cells treated with only Smokeless tobacco showed up-regulation of anti-apoptotic marker gene, BCL2 and the SCC4 cells which were treated with smokeless tobacco followed by treatment with Piperine showed down regulation of Bcl2 and up regulation of BAD and TP53 (Pro-apototic marker genes) (Figure 4, 8). Student’s t-test was performed for statistical analysis of the results of expression and P value was found to be statistically significant (p>0.05)
Figure 1: % Cell Viability Graph of Smokeless Tobacco (ST) Treated by SCC4
Figure 2: Dose and Time Dependent Kinetics of ST Followed Piperine Treatment on SCC4
Impact Factor (JCC): 2.8872 Index Copernicus Value (ICV): 3.0 Piperine Induces Down Regulation of BCL2 and up Regulation of Bad in 11 Smokeless Tobacco Induced Human Oral Squamous Cell Carcinoma (SCC4)
Figure 1: % Cell Viability Graph of smokeless tobacco (ST) treated SCC4 cell line. Cells were treated with .78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100 μg/ml of tobacco. Cell viability was monitored by MTT assay. The percentage of viability was calculated as the following formula: (viable cells) %= (OD of drug-treated sample/OD of untreated sample) ×100
Figure 2: Dose and time dependent kinetics of ST followed by piperine treatment on SCC4 cells using MTT assay. ST induced SCC4 cells were treated with 3.125, 6.25, 12.5, 25, 50,100, 200, 400 μg/ml of piperine for 24 h, 48 h, and 72 h. Cell viability was monitored by MTT assay. The percentage of viability was calculated as the following formula: (viable cells) %= (OD of drug-treated sample/OD of untreated sample) ×100.
RNA EXTRACTION
RNA was extracted from SCC4 cells treated with tobacco and tobacco followed with piperine.
Quantification and Qualification was done on Nanodrop and Gel electrophoresis respectively.
Figure 3: Gel Electrophoresis Analysis of RNA Isolated from ST and ST Followed by Piperine Treated SCC4 Cells
Figure 4 Figure 5 Figure 6 Figure 7 Figure 8
Figure 4,5,6 Gel electrophoresis analysis of ST induced followed by piperine treated SCC4 cells.Gel electrophoresis results showed specific amplification sequence of interest. Molecular marker size - 100 bp ladder, 123 bp PCR product of Bcl-2 gene: 249 bp PCR product of BAD gene: 133 bp PCR product of TP53 gene.
Figure 7,8 Gel electrophoresis analysis of ST treated SCC4 cells.Gel electrophoresis results showed specific amplification sequence of interest. Molecular marker size - 100 bp ladder, 123 bp PCR product of Bcl-2 gene: 249 bp PCR product of BAD gene: 133 bp PCR product of TP53 gene. www.tjprc.org [email protected] 12 Nikhil Khurana, Rajvardhan Singh, Anjana Singh & Vishwas Tripathi
EXPRESSION ANALYSIS OF ST AND ST FOLLOWED BY PIPERINE TREATED SCC4 CELL LINE
Figure 9 DISCUSSIONS
Despite the availability of several therapeutic options, a safer and more effective modality is urgently needed for treatment of head and neck cancer. Specific chemotherapy is effective, but severe side effects on normal cells and drug resistance by cancer cells limit its clinical use and underscore the need for unconventional therapies using less toxic substances. Natural products, derived from plants have become a key source of anti-cancer therapies. Evidence is accumulating that compounds of plant origin (phytochemical) exert anti-cancer effects with less toxicity. Black pepper, the spice of the millennia has been widely used in various food preparations throughout the globe. The average daily intake of black pepper in United State alone has been estimated at 359 mg. Piperine accounts for 5% to 9% of the black pepper content, implying the daily intake of approximately 60–110 µM. Piperine (trans-trans isomer of 1-piperoyl piperidine) is the active principle and the main ingredient of black pepper used as a traditional medicine in India. The potential of piperine has been has been demonstrated as anti-cancer agent in several literatures. However little is known about the beneficial effects of piperine against Head and neck cancer. One of the major causes of Head and neck cancer is the indiscriminate use of tobacco in its various forms. Tobacco has several a carcinogenic compound among which nitrosamine is the major one responsible for its carcinogenic effect. Besides tobacco also has nicotine which leads to its addiction to the users. Therefore, in this study we first treated cells with different dosages of smokeless tobacco and thereafter identified the effect of piperine on SCC4 head and neck cancer cells to observe whether piperine has anti-proliferative effect on smokeless tobacco induced human head and neck cancer cells. Our results of MTT assay clearly showed the proliferative effect of Tobacco and anti-proliferative effect of piperine in smokeless tobacco treated human head and neck cancer cells. Further in order to revalidate our result of MTT assay, we observed the expression of apoptotic and pro apoptotic marker genes. Our results clearly showed that piperine induces up-regulation of apoptotic marker genes (BAD and Tp53) and down regulation of anti-apoptotic marker gene BCL2.Our study has important implications. Based on our findings future studies may be conducted to elucidate the signaling pathways modulated by piperine in head and neck cancer.
REFERENCES
1. Centers for Disease Control and Prevention. Smoking & Tobacco Use. [Cited January 15, 2010]; Trends in Current Cigarette Smoking Among High School Students and Adults, United States, 1965–2007]. 2009.
Impact Factor (JCC): 2.8872 Index Copernicus Value (ICV): 3.0 Piperine Induces Down Regulation of BCL2 and up Regulation of Bad in 13 Smokeless Tobacco Induced Human Oral Squamous Cell Carcinoma (SCC4)
2. Centers for Disease Control and Prevention. MMWR Morb. Mortal. Wkly. Rep. Vol.58. 2009. Cigarette smoking among adults and trends in smoking cessation-United States, 2008; p. 1227-1232.
3. Shafey O, et al. The Tobacco Atlas. Atlanta, GA: American Cancer Society; 2009.
4. World Health Organization. Tobacco Key Facts. 2009. Retrieved from
http://www.who.int/topics/tobacco/facts/en/index.html
5. Warnakulasuriya, K.A., Ralhan, R (2007) Clinical, pathological, cellular and molecular lesions caused by oral smokeless tobacco--a review. J Oral Pathol Med 36: 63–77.
6. Cogliano, V., Straif, K., Baan, R., Grosse, Y., Secretan B., et al. (2004) Smokeless tobacco and tobacco-related nitrosamines. Lancet Oncol 5: 708.
7. Sapkota, A.R., Berger, S., Vogel, T.M. (2010) Human pathogens abundant in the bacterial metagenome of cigarettes. Environ Health Perspect 118: 351–356.
8. Mc Clave, A. K., Whitney, N., Thorne, S.L., Mariolis, P., Dube, S.R., (2010) Adult tobacco survey - 19 States, 2003-2007. MMWR Surveill Summ 59: 1–75.
9. International Agency for Research on Cancer. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Vol. v. 89. Lyon, FR: IARC; 2007. Smokeless tobacco and tobacco-specific nitrosamines; p. 41-417.
10. Hecht, S.S., (1988) Biochemistry, biology, and carcinogenicity of tobacco-specific N-nitrosamines. Chem. Res. Toxicol; 11:559–603. [Pub Med: 9625726]
11. Hecht, S.S., Hoffmann, D. (1988) Tobacco-specific nitrosamines, an important group of carcinogens in tobacco and tobacco smoke. Carcinogenesis; 9:875–884. [Pub Med: 3286030]
12. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Smokeless tobacco and some tobacco-specific N-nitrosamines. Lyon, France: International Agency for Research on Cancer; c2007. IARC monographs on the evaluation of carcinogenic risks to humans, Vol. 89.
13. Winstanley, M. (2009). The health effects of active smoking. In: Scollo MM, Winstanley MH, eds. Tobacco in Australia: Facts and Issues. 3rd ed. Melbourne: Cancer Council Victoria; http://www.tobaccoinaustralia.org.au/chapter-3-health-effects. Accessed January 22, 2009.
14. Xu, J., Huang, H., Pan, C., Zhang, B., Liu, X., Zhang, L. (2007) Nicotine inhibits apoptosis induced by cisplatin in human oral cancer cells. Int. J. Oral Maxillofac. Surg., 36, 739–744.
15. Kurinna, S.M., Tsao, C.C., Nica, A.F., Jiffar, T., Ruvolo, P.P., (2004) Ceramide promotes apoptosis in lung cancer-derived A549 cells by a mechanism involving c-Jun NH2-terminal kinase. Cancer Res 64: 7852–7856.
16. Zhang, J., Kamdar, O., Le, W., Rosen, G.D., & Upadhyay, D. (2009) Nicotine induces resistance to chemotherapy by modulating mitochondrial signaling in lung cancer. Am J Respir Cell Mol Biol 40: 135–146
17. Macha, M.A., Matta, A., Chauhan, S.S., Siu, K. W., Ralhan,R. (2011) Guggulsterone targets smokeless tobacco induced PI3K/Akt pathway in head and neck cancer cells. PLoS One. Feb 24; 6(2):e14728. Sakai, E., Tsuchida, N. (1992) most human squamous cell carcinomas in the oral cavity contain mutated p53 tumor-suppressor www.tjprc.org [email protected] 14 Nikhil Khurana, Rajvardhan Singh, Anjana Singh & Vishwas Tripathi
genes. Oncogene; 7: 927–933.
18. Kim, M.S., Li, S.L., Bertolami, C.N., Cherrick, H.M., Park, N.H.(1993) .
19. State of p53, Rb and DCC tumor suppressor genes in human oral cancer cell lines. Anticancer Res; 13: 1405–1413.
Impact Factor (JCC): 2.8872 Index Copernicus Value (ICV): 3.0