Blockade of PI3K/AKT pathway enhances sensitivity of Raji cells to chemotherapy through down-regulation of HSP70

Abstract

Up-regulation of heat shock protein 70 (HSP70) could be elicited primarily by heat in former studies, and this was proved to be associated with cancer progression. Burkitt's lymphoma is one of highly aggressive B-cell non-Hodgkin's lymphoma and is one of the fastest growing human tumors. To investigate the effect of HSP70 expression on the sensitivity of human Burkitt lymphoma cells (Raji cells) to chemotherapy and its role in the involvement of PI3K/AKT pathway, we evaluated the effects of LY294002, a PI3K inhibitor, on the expression of HSP70 and cell sensitivity to adriamycin (ADM) or cisplatin (DDP). In present study, expressions of HSP70, AKT and phosphorylated AKT (p-AKT) in Raji cells were measured by Western-Blot. Apoptosis index of Raji cells was examined by flow cytometry. Cytotoxicities of adriamycin (ADM) and cisplatin (DDP) were determined by WST-8 assay. We found that hyperthermia (42 degrees for 1 hour) up-regulated the expression of HSP70 expression and blockade of PI3K/AKT pathway down-regulated HSP70 expression in Raji cells. Compared to cells treated with ADM or DDP alone, hyperthermia protected cells from chemotherapy while LY294002 enhanced sensitivity of Raji cells to chemotherapy. Our results suggested down-regulation of HSP70 expression by blockade of PI3K/AKT pathway maybe responsible for the increased sensitivity of Raji cells to chemotherapy. Targeting PI3K/AKT pathway or inhibiting HSP70 expression may be beneficial for chemotherapy treatment of Burkitt lymphoma patients.

Figure 1

Effects of HT and LY294002 on expression of HSP70 and cell apoptosis. (A) Raji cells were cultured at 42.0°C for 1 hour and then they were returned to 37.0°C for the indicated time of recovery. The expression of HSP70 was detected by Western-Blot. (B) Apoptosis rate of Raji cells induced by LY294002 at indicated concentration was measured by FACS. (C) Raji cells were cultured at 42.0°C for 1 hour and then they were returned to 37.0°C for 8 hours or received LY294002 (20 μM) teratment for 8 hours. Expression of HSP70 and p-AKT in Raji cells were measured by Western-Blot.

Figure 2

Apoptosis induced by HT, LY294002 and DDP. (A) Apoptosis of Raji cells in HT + DDP(5 μg/ml) group; (B) Apoptosis of Raji cells in HT + DDP(10 μg/ml)group; (C) Apoptosis of Raji cells in DDP(5 μg/ml)group; (D) Apoptosis of Raji cells in DDP(10 μg/ml)group; (E) Apoptosis of Raji cells in LY294002 + DDP(5 μg/ml)group; (F) Apoptosis of Raji cells in LY294002 + DDP(10 μg/ml)group.

Figure 3

Effects of HT and LY294002 on ADM and DDP induced cytotoxicity. WST-8 assay was used to detect survival rates of Raji cells in indicated groups. Cell viability were inhibited by ADM in a concentration-dependent manner. Raji cells in HT + ADM group (RH gropup) showed more resistant to ADM than ADM group (R group). When treated with LY294002 and ADM (RL group), Raji cells were more sensitive to ADM (A). Similar phenomenon was also observed when Raji cells were treated with DDP (B).