Down-regulations of both HSP27 and TGF-β expression overcome the resistance of hepatocellular car-cinoma cells to Sorafenib

Abstract

Hepatocellular carcinoma (HCC) is one of the most common malignances worldwide. Although there have been developments in surgical strategies and percutaneous techniques, such as ethanol in-jection and radiofrequency ablation and transcatheter arterial che-moembolization (TACE), the overall outcome for HCC patients re-mains poor, as HCC is commonly detected at a late stage, when ther-apeutic options are limited. These patients show extremely poor prognoses, where the overall 5-year survival rate is poor. More than 630,000 patients die from HCC annually, representing the third highest number of cancer-related deaths. Therefore, it is necessary to improve anticancer therapies that effectively and specifically target liver tumor cells. Sorafenib, a multikinase inhibitor, is the standard therapy for patients with advanced-stage HCC. Although the results of sorafenib for patients with advanced HCC are encouraging, the benefits of sorafenib are limited by the low response rates of HCC cells, owing to a resistance to the treatment. Therefore, it is essential to develop more effective therapeutic strategies to overcome resistance and improve the efficacy of sorafenib in treating HCC patients. In this study, we tried to unravel the mechanism underlying the resistance of HCC cells to sorafenib via the development of a novel anti-cancer gene therapy by using two short hairpin RNAs (shRNAs) against heat shock protein 27 (HSP27) and transforming growth fac-tor-β (TGF-β) delivered by an adenovirus. We found that p38MAPkinase activity was inhibited by low concentrations of sorafenib, which could potentially lead to sorafenib resistance in HCC cell lines. Subsequently, we used constitutive form of MKK3/6(MKK3/6E) to confirm that massive cell death was induced by the activation of p38, which demonstrated the ability to activate p38 without any stimulation. In addition, sorafenib resistance was reduced by the activation of p38. Then we used shRNAs against TGF-β and HSP27 to increase the activity of p38 and overcome sorafenib resistance in HCC cell lines. TGF-β expression was decreased by treatment with high con-centration of sorafenib, thereby inducing effective cell death. Subse-quently, we confirmed that TGF-β shRNA effectively recovered the phosphorylation of p38 inhibited by sorafenib, and increased the sensitivity of HCC cells to sorafenib, thereby inducing cell death and overcoming the resistance of HCC cells to sorafenib. We also observed a HSP27 shRNA-induced reduction in the expression of HSP27, a protein involved in general resistance, which also enhanced the cell death response of HCC cells to sorafenib. There are many features of adenoviruses make them well suited for gene delivery, as recombinant adenoviruses can be grown to high titers, and have a relatively high capacity for transgene insertion, usually without the incorporation of viral DNA into the host cell ge-nome. The co-expression of both the shTGF-β and shHSP27 delivered by adenovirus, combined with low concerntration of sorafenib, effectively recovered p38 activity and the phosphorylation of HSP27 (p-HSP27), and resulted in a reduction in the levels of HSP27 protein, all of which synergistically lead to HCC cell death. Our study provides a new therapeutic strategy for HCC that not only overcomes the resistance of HCC to sorafenib, but also utilizes cutting edge of gene therapies.

간암(HCC)은 세계적으로 발병률이 높은 악성종양 중 하나이다. 현재 외과 수술과 고주파 열 치료 및 간동맥 화학 색전술 등이 간암 치료에 이용되고 있다. 하지만 HCC환자가 보통 말기로 발견되어 치료 방법의 선택이 제한되어 있고 예후도 좋지 않아서 5 년 생존율이 낮다. 해마다 630,000명 이상의 환자가 간암으로 사망하여, 암으로 인한 사망의 3위를 차지하고 있다. 따라서, 간암에 대해 효율적인 항암 요법을 개발하는 것이 필요하다. Sorafenib은 다중 인산화효소 억제제로 개발되어 말기 간암 치료제로 이용되고 있다. 말기 간암 환자에서 sorafenib의 치료 효과가 고무적이지만 많은 환자에서 치료 효과가 없거나 곧 저항성을 나타낸다. 따라서 말기 간암 환자에서 sorafenib의 효능을 개선하고 저항성을 극복할 수 있는 새로운 치료 전략을 개발하는 것이 필요하다. 본 연구에서는 HCC에서 sorafenib 약제 저항성을 극복하기위하여 adenovirus 전달체에 heat shock protein 27 (HSP27)과 transforming growth factor-β (TGF-β) mRNA를 타겟팅하는 short ha...