Effects of disulfiram combined with copper ions on proliferation and apoptosis of lenvatinib resistance Huh7 cells_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

DONG Bowen ¹ , LI Ziyi ¹ , LI Weidong ² ,  DAI Wenjie ³

1. Key Laboratory of The Ministry of Education of Hepatosplenic Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin 150001, P. R. China;
2. The Third Department of General Surgery, The Fourth Affiliated Hospital of Harbin Medical University, Harbin 150001, P. R. China;
3. Department of Thyroid Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin 150001, P. R. China;

Corresponding author：

DAI Wenjie, Email: davidhmu@163.com

Keywords：

hepatocellular carcinoma; disulfiram-Cu; lenvatinib resistance; p-Akt; caspase-9

DOI：

10.7507/1007-9424.201906108

Video：

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Obejective To establish the lenvatinib-resistant model of hepatocellular carcinoma (HCC) in vitro in order to study the effect of disulfiram combined with copper ions on the proliferation and apoptosis of lenvatinib-resistant Huh7 cells.Methods The half-inhibitory concentration (IC₅₀) of Huh7 cells treated with lenvatinib was detected by the CCK8 method, and the resistance of Huh7 cells to lenvatinib was induced by increasing concentration method. The cell treatment of each group: The Huh7 cells cultured with common culture medium and with 10 μmol/L lenvatinb in the control group and lenvatinib sensitive group, respectively; The lenvatinib-resistant Huh7 cells cultured with 10 μmol/L lenvatinb, with 10 μmol/L lenvatinib+1 μmol/L disulfiram+0.5 μmol/L copper ions, and with 10 μmol/L lenvatinib+10 μmol/L Akt inhibitor MK2206 in the lenvatinib-resistant group, combined drugs group, and inhibitor group, respectively. The cell survival and apoptosis rates were detected by the CCK8 and flow cytometry, respectively. The Western blot method was used to detect the expressions of phospho-protein kinase B (p-Akt), cysteine aspartic acid specific protease (caspase-9), and B-cell lymphoma-2 (Bcl-2) proteins. The Transwell assay was used to detect the cell invasiveness.Results The lenvatinib-resistant cell line was successfully established. The concentrations of 10, 20, 40, 60, 80 and 160 μmol/L leventinib were applied to the Huh7 cells for 48 h, and the IC₅₀ was calculated to be 12.35 μmol/L. The cell survival rate of the lenvatinib-resistant group was significantly higher than that of the lenvatinib sensitive group (P<0.01), which in the combined drugs group was significantly lower than that of the lenvatinib-resistant group (P<0.01), which in the inhibitor group was significantly lower than that of the lenvatinib-resistant group (P<0.05). The apoptosis rate of the combined drugs group was significantly higher than that of the lenvatinib-resistant group (P<0.01). Compared with the lenvatinib-resistant group, the expression of p-Akt protein was significantly decreased (P<0.01) and caspase-9 expression was significantly increased (P<0.01) in the combined drugs group, while the difference of Bcl-2 protein between the lenvatinib-resistant group and the combined drugs group was not statistically significant (P>0.05). The invasive cells were more in the combined drugs group as compared with the lenvatinib-resistant group (P<0.01). Conclusion The combination of disulfiram and copper ion could increase the sensitivity of lenvatinib to lenvatinib-resistant Huh7 cells, the cell inhibition rate is significantly increased, it’s mechanism might be related to the inhibiting p-Akt expression and promoting caspase-9 expression.

Citation： DONG Bowen, LI Ziyi, LI Weidong, DAI Wenjie. Effects of disulfiram combined with copper ions on proliferation and apoptosis of lenvatinib resistance Huh7 cells. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2020, 27(2): 168-172. doi: 10.7507/1007-9424.201906108 Copy

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2.	Llovet JM, Di Bisceglie AM, Bruix J, et al. Design and endpoints of clinical trials in hepatocellular carcinoma. J Natl Cancer Inst, 2008, 100(10): 698-711.
3.	de Rosamel L, Blanc JF. Emerging tyrosine kinase inhibitors for the treatment of hepatocellular carcinoma. Expert Opin Emerg Drugs, 2017, 22(2): 175-190.
4.	Glen H, Mason S, Patel H, et al. E7080, a multi-targeted tyrosine kinase inhibitor suppresses tumor cell migration and invasion. BMC Cancer, 2011, 11: 309.
5.	Spallanzani A, Orsi G, Andrikou K, et al. Lenvatinib as a therapy for unresectable hepatocellular carcinoma. Expert Rev Anticancer Ther, 2018, 18(11): 1069-1076.
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8.	Ikuta K, Yano S, Trung VT, et al. E7080, a multi-tyrosine kinase inhibitor, suppresses the progression of malignant pleural mesothelioma with different proangiogenic cytokine production profiles. Clin Cancer Res, 2009, 15(23): 7229-7237.
9.	Ogino H, Hanibuchi M, Kakiuchi S, et al. E7080 suppresses hematogenous multiple organ metastases of lung cancer cells with nonmutated epidermal growth factor receptor. Mol Cancer Ther, 2011, 10(7): 1218-1228.
10.	Chen D, Cui QC, Yang H, et al. Disulfiram, a clinically used anti-alcoholism drug and copper-binding agent, induces apoptotic cell death in breast cancer cultures and xenografts via inhibition of the proteasome activity. Cancer Res, 2006, 66(21): 10425-10433.
11.	Allensworth JL, Evans MK, Bertucci F, et al. Disulfiram (DSF) acts as a copper ionophore to induce copper-dependent oxidative stress and mediate anti-tumor efficacy in inflammatory breast cancer. Mol Oncol, 2015, 9(6): 1155-1168.
12.	Choi SA, Choi JW, Wang KC, et al. Disulfiram modulates stemness and metabolism of brain tumor initiating cells in atypical teratoid/rhabdoid tumors. Neuro Oncol, 2015, 17(6): 810-821.
13.	Kim JY, Cho Y, Oh E, et al. Disulfiram targets cancer stem-like properties and the HER2/Akt signaling pathway in HER2-positive breast cancer. Cancer Lett, 2016, 379(1): 39-48.
14.	Llovet JM, Fuster J, Bruix J. Intention-to-treat analysis of surgical treatment for early hepatocellular carcinoma: resection versus transplantation. Hepatology, 1999, 30(6): 1434-1440.
15.	Mazzaferro V, Regalia E, Doci R, et al. Liver transplantation for the treatment of small hepatocellular carcinomas in patients with cirrhosis. N Engl J Med, 1996, 334(11): 693-699.
16.	Chiba T, Suzuki E, Yuki K, et al. Disulfiram eradicates tumor-initiating hepatocellular carcinoma cells in ROS-p38 MAPK pathway-dependent and -independent manners. PLoS One, 2014, 9(1): e84807.
17.	Nechushtan H, Hamamreh Y, Nidal S, et al. A phase Ⅱb trial assessing the addition of disulfiram to chemotherapy for the treatment of metastatic non-small cell lung cancer. Oncologist, 2015, 20(4): 366-367.
18.	Zhang H, Chen D, Ringler J, et al. Disulfiram treatment facilitates phosphoinositide 3-kinase inhibition in human breast cancer cells in vitro and in vivo. Cancer Res, 2010, 70(10): 3996-4004.
19.	Loo TW, Clarke DM. Blockage of drug resistance in vitro by disulfiram, a drug used to treat alcoholism. J Natl Cancer Inst, 2000, 92(11): 898-902.
20.	Chen KF, Chen HL, Tai WT, et al. Activation of phosphatidylinositol 3-kinase/Akt signaling pathway mediates acquired resistance to sorafenib in hepatocellular carcinoma cells. J Pharmacol Exp Ther, 2011, 337(1): 155-161.
21.	Gulhati P, Zaytseva YY, Valentino JD, et al. Sorafenib enhances the therapeutic efficacy of rapamycin in colorectal cancers harboring oncogenic KRAS and PIK3CA. Carcinogenesis, 2012, 33(9): 1782-1790.
22.	Hennessy BT, Smith DL, Ram PT, et al. Exploiting the PI3K/AKT pathway for cancer drug discovery. Nat Rev Drug Discov, 2005, 4(12): 988-1004.
23.	Zhang PF, Li KS, Shen YH, et al. Galectin-1 induces hepatocellular carcinoma EMT and sorafenib resistance by activating FAK/PI3K/AKT signaling. Cell Death Dis, 2016, 7: e2201.
24.	Desai JR, Ochoa S, Prins PA, et al. Systemic therapy for advanced hepatocellular carcinoma: an update. J Gastrointest Oncol, 2017, 8(2): 243-255.
25.	Ikeda K, Kudo M, Kawazoe S, et al. Phase 2 study of lenvatinib in patients with advanced hepatocellular carcinoma. J Gastroenterol, 2017, 52(4): 512-519.

1. Villanueva A, Llovet JM. Targeted therapies for hepatocellular carcinoma. Gastroenterology, 2011, 140(5): 1410-1426.
2. Llovet JM, Di Bisceglie AM, Bruix J, et al. Design and endpoints of clinical trials in hepatocellular carcinoma. J Natl Cancer Inst, 2008, 100(10): 698-711.
3. de Rosamel L, Blanc JF. Emerging tyrosine kinase inhibitors for the treatment of hepatocellular carcinoma. Expert Opin Emerg Drugs, 2017, 22(2): 175-190.
4. Glen H, Mason S, Patel H, et al. E7080, a multi-targeted tyrosine kinase inhibitor suppresses tumor cell migration and invasion. BMC Cancer, 2011, 11: 309.
5. Spallanzani A, Orsi G, Andrikou K, et al. Lenvatinib as a therapy for unresectable hepatocellular carcinoma. Expert Rev Anticancer Ther, 2018, 18(11): 1069-1076.
6. Kudo M. A new era of systemic therapy for hepatocellular carcinoma with regorafenib and lenvatinib. Liver Cancer, 2017, 6(3): 177-184.
7. Kudo M. Lenvatinib may drastically change the treatment landscape of hepatocellular carcinoma. Liver Cancer, 2018, 7(1): 1-19.
8. Ikuta K, Yano S, Trung VT, et al. E7080, a multi-tyrosine kinase inhibitor, suppresses the progression of malignant pleural mesothelioma with different proangiogenic cytokine production profiles. Clin Cancer Res, 2009, 15(23): 7229-7237.
9. Ogino H, Hanibuchi M, Kakiuchi S, et al. E7080 suppresses hematogenous multiple organ metastases of lung cancer cells with nonmutated epidermal growth factor receptor. Mol Cancer Ther, 2011, 10(7): 1218-1228.
10. Chen D, Cui QC, Yang H, et al. Disulfiram, a clinically used anti-alcoholism drug and copper-binding agent, induces apoptotic cell death in breast cancer cultures and xenografts via inhibition of the proteasome activity. Cancer Res, 2006, 66(21): 10425-10433.
11. Allensworth JL, Evans MK, Bertucci F, et al. Disulfiram (DSF) acts as a copper ionophore to induce copper-dependent oxidative stress and mediate anti-tumor efficacy in inflammatory breast cancer. Mol Oncol, 2015, 9(6): 1155-1168.
12. Choi SA, Choi JW, Wang KC, et al. Disulfiram modulates stemness and metabolism of brain tumor initiating cells in atypical teratoid/rhabdoid tumors. Neuro Oncol, 2015, 17(6): 810-821.
13. Kim JY, Cho Y, Oh E, et al. Disulfiram targets cancer stem-like properties and the HER2/Akt signaling pathway in HER2-positive breast cancer. Cancer Lett, 2016, 379(1): 39-48.
14. Llovet JM, Fuster J, Bruix J. Intention-to-treat analysis of surgical treatment for early hepatocellular carcinoma: resection versus transplantation. Hepatology, 1999, 30(6): 1434-1440.
15. Mazzaferro V, Regalia E, Doci R, et al. Liver transplantation for the treatment of small hepatocellular carcinomas in patients with cirrhosis. N Engl J Med, 1996, 334(11): 693-699.
16. Chiba T, Suzuki E, Yuki K, et al. Disulfiram eradicates tumor-initiating hepatocellular carcinoma cells in ROS-p38 MAPK pathway-dependent and -independent manners. PLoS One, 2014, 9(1): e84807.
17. Nechushtan H, Hamamreh Y, Nidal S, et al. A phase Ⅱb trial assessing the addition of disulfiram to chemotherapy for the treatment of metastatic non-small cell lung cancer. Oncologist, 2015, 20(4): 366-367.
18. Zhang H, Chen D, Ringler J, et al. Disulfiram treatment facilitates phosphoinositide 3-kinase inhibition in human breast cancer cells in vitro and in vivo. Cancer Res, 2010, 70(10): 3996-4004.
19. Loo TW, Clarke DM. Blockage of drug resistance in vitro by disulfiram, a drug used to treat alcoholism. J Natl Cancer Inst, 2000, 92(11): 898-902.
20. Chen KF, Chen HL, Tai WT, et al. Activation of phosphatidylinositol 3-kinase/Akt signaling pathway mediates acquired resistance to sorafenib in hepatocellular carcinoma cells. J Pharmacol Exp Ther, 2011, 337(1): 155-161.
21. Gulhati P, Zaytseva YY, Valentino JD, et al. Sorafenib enhances the therapeutic efficacy of rapamycin in colorectal cancers harboring oncogenic KRAS and PIK3CA. Carcinogenesis, 2012, 33(9): 1782-1790.
22. Hennessy BT, Smith DL, Ram PT, et al. Exploiting the PI3K/AKT pathway for cancer drug discovery. Nat Rev Drug Discov, 2005, 4(12): 988-1004.
23. Zhang PF, Li KS, Shen YH, et al. Galectin-1 induces hepatocellular carcinoma EMT and sorafenib resistance by activating FAK/PI3K/AKT signaling. Cell Death Dis, 2016, 7: e2201.
24. Desai JR, Ochoa S, Prins PA, et al. Systemic therapy for advanced hepatocellular carcinoma: an update. J Gastrointest Oncol, 2017, 8(2): 243-255.
25. Ikeda K, Kudo M, Kawazoe S, et al. Phase 2 study of lenvatinib in patients with advanced hepatocellular carcinoma. J Gastroenterol, 2017, 52(4): 512-519.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Effects of disulfiram combined with copper ions on proliferation and apoptosis of lenvatinib resistance Huh7 cells

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