Effects Of IL-24 Combined With Autophagy Inhibitor On The Growth Of Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma(OSCC) is one of the most common malignant tumors of oral cavity. OSCC is highly aggressive, it often damage the surrounding tissues like nerves, glands, muscles and bones, and cause lymph node metastasis in it,s early stage. Conventional treatments including surgical removal, chemotherapy and radiotherapy. Although surgery can removal of tumor, it has many disadvantages. Surgical removal often cause severe tissue defect and affect the patient’s physical and mental health seriously. For chemotherapy and radiotherapy,besides killing tumor cells, they also damage the normal cells and cause complications of bone marrow suppression, liver and kidney damage, damage of immune system and even death. Hence the effect of conventional treatments, such as surgical removal, chemotherapy and radiotherapy is limited for OSCC.Therefore, a new effective way to cure OSCC is in urgent need.With the development of molecular biology, gene therapy provides a new way for cancer therapy. IL-24 is a new type of tumor specificity apoptosis inducing factor. IL-24 transfection leads to apoptosis of tumor cells, inhibition of the growth of blood vessels in tumors and makes tumor cells more sensitive to chemotherapy and radiotherapy. Moreover, IL-24 transfection can makes the body inducing immune responses with an anti tumor reactions and it is surprising that,IL-24 does not have any harmful effects on normal cells. But it also found that,similar to radiotherapy and chemotherapy, tumor cells often exhibit resistance to IL-24 treatment. The therapy resistance of tumor cells is one of the important reasons for tumor recurrence and it is the main reason for the failure of non-operative treatment. It is reported that, when the tumor cells respond to radiotherapy and chemotherapy, autophagy is activated and contributes to maintaining homeostasis through degradation of impaired or unnecessary macro-molecules and organelles, thereby providing energy to cancer cells.Autophagy helps cancer cells to escape the damage caused by radiation and chemotherapy. Therefore, as one of the mechanisms of drug resistance, autophagy has been widely concerned, and people attempt to improve the effectiveness of cancer treatments through adjusting autophagy.Although IL-24 has a strong inhibitory effect on a variety of malignant tumors, there is no relevant reports about IL-24 inhibiting the growth of OSCC and if OSCC cell exhibit resistence to IL-24 treatment. In order to explore the feasibility of IL-24 therapy for OSCC, in this study the effective concentration ofIL-24, the characteristics of intracellular expression and the mechanism of inhibiting growth of OCSS cells of IL-24 were analyzed systematically. In addition to this,the induction of autophagy induced by IL-24 in OSCC cells and OSCC cell xenografts was studied. Besides, in this study the efficacy of IL-24 combined with autophagy inhibitor 3-MA for inhibiting the proliferation and growth of OSCC were investigated. For experimental procedure, first we confirmed the effective concentration of the new hybrid virus vector Ad LTR2EF1 and the recombinant Ad LTR2EF1α-IL-24, and then the gene expression ability and patterns of recombinant protein were analysed after infection of Ad LTR2EF1α-IL-24. We found that Ad LTR2EF1α-IL-24 could effectively express IL-24 proteins of two types, which were intracellular retention type and secretory type, in OSCC cells. After that, Ad LTR2EF1α-IL-24 was used to infect one of the OSCC cell line- KB cells and a spontaneously transformed aneuploid immortal keratinocyte cell line from adult human skin called Ha Ca T cells are used as control. We find that IL-24 can effectively inhibit the growth of KB cells and KB cell xenografts. To our surprise, IL-24 has no harmful effect on Ha Ca T cells and normal organs like liver, heart and lungs of tumor bearing nude mice. These results indicated that IL-24 could effectively inhibit the proliferation and growth of OSCC cells without significant toxicity to normal cells.To explore the possible mechanism of it, the changes of cell cycle, the apoptosis rate, expression and acivity of apoptosis related proteins of OSCC cells were detected. The results showed that IL-24 could enhance the expression and activity of apoptosis related proteins in OSCC cells, and thereby enhancing the apoptosis and thus inhibiting the proliferation and growth of it.In addition to this, through TEM observation, MDC and GFP-LC3 staining,and detection of autophagy related proteins we find that IL-24 enhance the autophagy of KB cells. This indicates that KB cells can produce resistance to IL-24 through autophagy, thus affecting the effect of IL-24 on KB cells.In order to overcome the obstacle of resistance to IL-24 and to identify whether autophagy inhibition can improve the effect of IL-24 on OSCC, a well known autophagy inhibitor 3-Methyladenine(3-MA) was introduced and the changes of cell cycle, the apoptosis rate, expression and acivity of apoptosis related proteins of OSCC cells were analysed after combined treatment of IL-24 with 3-MA. To our great surprise, the application of autophagy inhibitor 3-MA can significantly increase the effects of IL-24 on OSCC.To be concluded, through different methods and from different aspects we proved that IL-24 has a significant anticancer effect towards OSCC and discoverthat the main mechanism of IL-24 inhibiting the growth of OSCC is promoting the apoptosis of OSCC cells. In addition to this, we also found that IL-24 can induce autophagy in OSCC cells and autophagy inhibition can greatly improve the anticancer effects of IL-24 towards OSCC. Therefore, we believe that the application of combination of IL-24 and autophagy inhibitor will open up new perspectives in OSCC treatment.