INTRODUCTION:

Cancer are described by uncontrolled cell growth. In the world colorectal cancer (CRC) is been categorized as third most common cancer. The fourth most common reason of cancer-related mortality, with 0.7 million deaths per year, exceeded only by lung carcinoma, Heptocarcinoma and Gastrointestinal carcinoma¹. Tolvaptan are the oral drug which comes under the vasopressin Receptor Antagonist. It is used to treatment of hypervolemia and low sodium concentration in the blood patients with heart failure, the syndrome of inappropriate antidiuretic hormone secretion (SIADH and liver cirrhosis². In humans, vasopressin hormone acts on v1and v2 receptor. Tolvaptan blocks the v2 receptor. This drug inhibits the mitogenic effects of vasopressin on its receptors³. Vasopressin Receptor Antagonist has a Cytochome – P450 enzyme inhibit effect. The cell proliferation arrest and cell death of Hep cell line was proven in preview, which illustrations that tolvaptan has a role in inhibiting the hepatocyte². This orally active V2 receptor inhibitors are efficacious and well tolerated. Presently, most of the medications used in the management of cancer are cytotoxic. In specific, cytotoxic drugs are not only targets the cancer cells but also affect the fastest growing normal cells⁴. Therefore they may be unsafe to the human body. Newer drugs are designed in such a way to meet the need that are more particular to tumor cells. Studies have been reported that vasopressin antagonist have anticancer property on Hepatocytes⁵. This study shows that tolvaptan. There are very few drugs available in the market, for the colon cancer cell.

Tolvaptan a is V₂receptor antagonist approved by FDA for clinically significant hypervolemia and euvolemic hyponatremia. Expert panels have yet to reach a compromise regarding the appropriate use of V₂ receptor antagonists. Tolvaptan has decreases the chance of colon cancer in rats when given at therapeutic dose⁶. Cardiac protective effects of tolvaptan is seen in phase-2 trials⁷. Tolvaptan regulates the aquaporin expression, vasopressin decrease significantly in tolvaptan treated group. In a study they found out the upregulation of AQP-2 in cirrhotic rats in distal colon is found. It was found out that decrease in water reabsorption and diarrhea of Tolvaptan is mainly due the inhibition gene expression. Known adverse effects of these drugs are vomiting, nausea, headache and liver damage, therefore, administration of tolvaptan is limited to one month⁶.

Imatinib was the first protein kinase inhibitor considered to target a driver alteration in a cancer and it was accepted by FDA in 2001. Imatinib targets the BCR-ABL tyrosine kinase fusion protein. It is well absorbed after oral administration and reaches maximal plasma concertation. Imatinib –resistant ABL mutation were revealed in 2002 and led to the development of next –generation inhibitors, which may overcome the resistance. Imatinib has high –throughput screening against the BCR-ABL kinase. Imatinib is a BCR-ABL inhibitor, also inhibits the Src kinase binds to the open (active) and close inactive alignment of the BCR-ABL.it is more effect for gastro intestinal carcinoma¹³. Hence it is used has positive control.

AIM:

To elucidate the anticancer activate using invitro -MTT assay of Tolvaptan in comparison with imatinib on colorectal cell lines.

CELL LINE AND CULTURE:

HT-29 cell line was purchased from Veterinary College, Vepery, Chennai. To maintain the cells alive, the cells were preserved in Minimal Essential Medium supplemented with Fetal bovine serum FBS 10%, penicillin (100U/ml), and streptomycin (100μg/ml) in a humidified atmosphere of 50μg/ml CO2 at 37°C.

REAGENTS:

MEM was purchased from Hi Media Laboratories Fetal Bovine Serum (FBS) was obtained from Cistron laboratories Trypsin, methylthiazolyl diphenyl- tetrazolium bromide (MTT) and Dimethyl sulfoxide (DMSO) were purchased from (Sisco research laboratory chemicals Mumbai). All of other chemicals and reagents were purchased from Sigma Aldrich Mumbai¹¹.

“In Vitro assay for Anticancer activity (MTT assay) (Mossman, 1983)

Cells (1 × 105/well) were plated in 24-well plates and incubated in 37 C with 5% CO₂ condition. After the cell ranges the confluence, the different concentrations of the samples were added and incubated for 24hrs. After incubation, the sample was removed from the well and washed with phosphate-buffered saline (pH7.4) or MEM deprived of serum. 100µl/well (5mg/ml) of 0.5% 3(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl--tetrazolium bromide (MTT) was added and incubated for 4 hours. After incubation, 1ml of DMSO was added in all the wells. The absorbance at 570nm was measured with UV- Spectrophotometer using DMSO as the blank. Measurements were performed and the concentration required for a 50% inhibition (IC50) was determined graphically¹².

The % cell viability was calculated using the following formula:

A570 of treated cells

% cell viability = –––––––––––––––––– × 100”

A570 of control cells

Graphs are plotted using the % of Cell Viability at Y-axis and concentration of the sample in X axis. Cell control and sample control is included in each assay to compare the full cell viability assessments.

RESULT:

At concentrations of 7.8, 15.6, 31.2, 62.5, 125, 250, 500, 1000μg/ml, the cell viability of Tolvaptan was 64.77%, 61.77%, 53.69%, 39.20%, 25.85%, 11.36%, 5.39% and 1.42% respectively as shown in [Table-1and Figure-1). Hence, the half maximal inhibitory concentration of Tolvaptan was at the concentration of 31.2μg/ml. The anticancer effect of tolvaptan on colon cancer cell lines at various toxicities is shown in [Figure-2]. At concentrations of 7.8, 15.6, 31.2, 62.5, 125, 250, 500, 1000μg/ml, the cell viability of imatinib was 67.61%, 65.05%, 57.67%, 48.01%, 30.96%, 16.76%, 5.96% and 2.55% respectively as shown in (Table2 and Figure-3). Hence, the half maximal inhibitory concentration of imatinib was at the concentration of 62.5μg/ml. The anticancer effect of tolvaptan on colon cancer cell lines at various toxicities is shown in (Figure-4)

The anticancer effect of tolvaptan and Imatinib when treated on HT-29 cell line starting minimum to maximum dose concentration (µg/ml), the percentage of cell viability was seen.

Table-1: Anticancer effect of Tolvaptan on colon cancer cell line.

S. No	Concentration (µg/ml)	Dilutions	Absorbance (O.D)	Cell viability (%)
1	1000	Neat	0.005	1.42
2	500	1:1	0.019	5.39
3	250	1:2	0.040	11.36
4	125	1.4	0.091	25.85
5	62.5	1:8	0.138	39.20
6	31.2	1.16	0.189	53.69
7	15.6	1.32	0.215	61.07
8	7.8	1:64	0.228	64.77
9	Cell control	-	0.352	100

Figure-1: Anticancer effect of Tolvaptan on colon cancer cell line graph.

a) Normal HT-29 Cell line b) Toxicity- 1000µg/ml

b)Toxicity- 31.2µg/ml d)Toxicity- 7.8 µg/ml

[Fig-2 a-d]: Anticancer effect of Tolvaptan on colon cancer cell line.

a) Normal Colon Cancer Cell line, b) Cancer cell line at Toxicity- 1000μg/ml, c) Cancer cell line at Toxicity- 31.2μg/ml,

d) Cancer cell line at Toxicity –7.8 μg/ml

Table-2: Anticancer effect of Imatinib on colon cancer cell line.

S. No	Concentration (µg/ml)	Dilutions	Absorbance (O.D)	Cell viability(%)
1	1000	Neat	0.005	2.55
2	500	1:1	0.019	5.96
3	250	1:2	0.040	16.76
4	125	1.4	0.091	30.96
5	62.5	1:8	0.138	48.01
6	31.2	1.16	0.189	57.67
7	15.6	1.32	0.215	65.05
8	7.8	1:64	0.228	67.61
9	Cell control	-	0.352	100

Fig-3: Anticancer effect of Imatinib on colon cancer cell line graph

Normal HT-29 Cell line Toxicity- 1000µg/ml

Toxicity- 62.5µg/ml Toxicity- 7.5 µg/ml

[Fig-4 a-d]: Anticancer effect of imatinib on colon cancer cell line.

a) Normal Colon Cancer Cell line, b) Cancer cell line at Toxicity- 1000μg/ml, c) Cancer cell line at Toxicity- 31.2μg/ml,

d) Cancer cell line at Toxicity –7.8μg/ml

Hence the percentage of maximum cell viability of tolvaptan is 64.77% (minimum cell inhibition 35.23%) observed at 7.9(µg/ml) whereas the minimum cell viability of tolvaptan is 1.42% (maximum cell inhibition is 98.58%) observed at 1000(µg/ml, In Imatinib Maximum cell viability is 67.61% (minimum cell inhibition-32.39%) and minimum cell viability is 2.55 % (maximum cell inhibition -97.45) Tolvaptan inhibited cell growth of HT-29 cells were treated with tolvaptan (0–100μM) for 24 h.

DISCUSSION:

Tolvaptan, a vasopressin antagonist which was approved in 2009 for the treatment of hyponatremia by the U.S. FDA. Studies have been focuses on the metabolic profile and pharmacological effects of tolvaptan. Consequently, liver impairment is a high risk which is association with tolvaptan was found⁶. The primary analysis in our study is to evaluate the anticancer effect of tolvaptan by HT-29 cell line. We found that cell growth of HT-29 cell line is inhibited by tolvaptan.

Previous studies shows Vasopressin have substantial anticancer action against hepatocytes. To elucidate the anticancer effects against the colon cancer. It was found that tolvaptan shows an anticancer activity in human HepG2 cells⁸. The underlying mechanisms of cytotoxicity induced by tolvaptan on the cell growth, cell cycle progression, DNA damage, and apoptosis is been examined⁹.

In addition, it was determined the role of mitogen-activated protein kinase (MAPK) activation, proteasomal degradation, and autophagy in these developments¹⁰. Tolvaptan has an anticancer effect starting from low to high concentrations when compared with the (Imanitib) positive control using MTT assay.

Imanitib an anticancer drug which is used as a positive control. An evaluation of the effect of tolvaptan on cell growth showed similar IC₅₀ values among HT-29 cell line. Hence, Tolvaptan has equally potent as Imatinib on HT-29 tumor cell lines.

CONCLUSION:

In our study, the anticancer effect of tolvaptan has been proved to be equally beneficial as imatinib in colon cancer cell lines. Vasopressin receptor blocker acts as a significant cytotoxic agent in various tumor conditions. To prove the other effects of this drug further studies are required.

ACKNOWLEDGEMENT:

The authors are grateful to our institute for providing all facilities Central Research and facilities in Sri Ramachandra Medical college and RI.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

REFERENCE:

1. Inés Mármol, Cristina Sánchez-de-Diego, Alberto Pradilla Dieste,Elena Cerrada, and María Jesús Rodriguez Yoldi Colorectal Carcinoma: A General Overview and Future Perspectives in Colorectal Cancer; Int J Mol Sci. 2017 Jan; 18(1): 197.

2. Jeffrey William J. Brock Sharin E. Roth Susan E. Shoaf Kim L.R. Brouwer Rachel Church Tom N. Grammatopoulos Linsey Stiles Scott Q. Siler Brett A. Howellpplication of a Mechanistic Model to Evaluate Putative Mechanisms of Tolvaptan Drug-Induced Liver Injury and Identify Patient Susceptibility Factors Toxicological Sciences, Volume 155, Issue 1, January 2017, Pages 61–74.

3. Miyazaki T, Fujiki H, Yamamura Y, Nakamura S, Mori T. Tolvaptan, an orally active vasopressin V(2)-receptor antagonist - pharmacology and clinical trials. Cardiovasc Drug Rev 2007; 25(1):1-13.

4. Robert W. Schrier, M.D., Peter Gross, M.D., Mihai Gheorghiade, M.D., Tomas Berl, M.D., Joseph G. Verbalis, M.D., Frank S. Czerwiec, M.D., Ph.D., and Cesare Orlandi, M.D; Tolvaptan, a Selective Oral Vasopressin, V2-Receptor, Antagonist, for Hyponatremia. N Engl J Med 2006; 355:2099-2112.

5. S. Garcha, Apurv Khanna; Review of Tolvaptan in the Treatment of Hyponatremia, Amarinder, Clinical Medicine Insights: Therapeutics 2011; 3: 315–325.

6. Laurence L. Brunton, Randa Hilal-Dandan, Björn C. Knollmann, Goodman & Gilman's: Modulation of Pulmonary, renal and cardiovascular function The Pharmacological Basis of Therapeutics 2017, 13e; Chapter 25, 468.

7. L. V. Rubinstein R. H. Shoemaker K. D. Paull R. M. Simon S. Tosini P. Skehan D. A. Scudiero A. Monks M. R. Boyd, JNCI, Comparison of In Vitro Anticancer-Drug-Screening Data Generated With a Tetrazolium Assay Versus a Protein Assay Against a Diverse Panel of Human Tumor Cell Lines,: Journal of the National Cancer Institute 1990, 82, 13,1113 –1117.

8. Yuanfeng Wu, Frederick A. Beland, Si Chen, Fang Liu, Lei Guo, Jia-Long Fang. "Mechanisms of tolvaptan-induced toxicity in HepG2 cells". Biochemical Pharmacology 2015; 95(4): 324-336.

9. Nida Iqbal and Naveed Iqbal, Review Article, Imatinib: A Breakthrough of Targeted Therapy in Cancer, Chemotherapy Research and Practice Volume 2014; 9.

10. Ling Gao Li Wang, Zhen Sun, Haiyan Li, Qiaoping Wang, Cheng Yi, Xiujie Wang. Morusin shows potent antitumor activity for human hepatocellular carcinoma in vitro and in vivo through apoptosis induction and angiogenesis inhibition. Drug Des Devel Ther 2017; 11: 1789–1802.

11. Johan van Meerloo, Gertjan J. L. Kaspers, Jacqueline Cloo, Cell Sensitivity Assays: The MTT Assay; Cancer Cell Culture; 237-245

12. C.A Amritha, Punnagai Kumaravelu, D. Darling Chellathai, Evaluation of Anticancer Effects of DPP-4 Inhibitors in Colon Cancer- An Invitro Study, Journal of Clinical and Diagnostic Research. 2015; 9(12);14-16.

13. Laurence L. Brunton, Randa Hilal-Dandan, Björn C. Knollmann, Goodman & Gilman's: Pharmacotherapy of neoplastic Disease; The Pharmacological Basis of Therapeutics 2017, 13e; Chapter 67,1213.

Received on 01.06.2019 Modified on 31.07.2019

Research J. Pharm. and Tech. 2020; 13(1):77-80.

DOI: 10.5958/0974-360X.2020.00014.1