The Experimental Study On The Heat Shock Protein 90 Inhibitor Ganetespib To Prevent Local Recurrence Of Hepatocellular Carcinoma After Microwave Ablation

Objective1. To investigate the effect of the novel HSP90 inhibitors Ganetespib on human liver cancer cell lines HepG2, to observe proliferations or apoptosis of HepG2 after drugs through experiments in vitro, and to verify the influence of different temperature of HSP90 expression level.2. To investigate the effect of heat shock protein90 (HSP90) modulation on tumour coagulation by combining microwave ablation with adjuvant Ganetespib in a rat tumour model, and to evaluate the toxic effect of Ganetespib.3. To investigate the effect of IV HSP90 inhibitor--Ganetespib--on tumor growth and endpoint survival when combined with microwave ablation in a rat tumor model.Methods1. CCK8 assay was applied to measure the optical density of different groups after different concentrations of Ganetespib with HepG2 cell line to calculate cell growth inhibition rate, Annexin V-FITC/PI double staining was to detect apoptosis rate of HepG2 cell line after Ganetespib, and ELISA to detect HSP90 expression levels after different temperature change.2. A Female mice bearing HepG2 xenografts received a single intravenous (i.v.) dose of the highest non-severely toxic dose (Ganetespib,150 mg/kg). Subsequently, the organs, including liver, spleen, lung and kidney were removed and fixed in formalin solution (10%). Histological study was employed to analyze the toxic effect.40 tumours were randomised into four groups:(1) MWA alone, (2) intravenous (IV) Ganetespib alone (75mg/kg), (3) IV Ganetesib 2 h before MWA, (4) Controlled group. Animals were sacrificed 24 h post-treatment and gross coagulation diameters were compared. Next, immunohistochemistry staining was performed for Hsp90 and cleaved caspase-3 expression.3. Twenty mice bearing HepG2 xenografts were used in this study. They were randomized into four experimental groups:(1) MWA alone, (2) intravenous (IV) Ganetespib alone (75mg/kg), (3) IV Ganetesib 2 h before MWA, (4) Controlled group. Kaplan-Meier survival analysis was performed using a tumor diameter of 2.5 cm or surviving more than 40 days as the defined survival endpoint.Results1. The results of CCK8 showed that Ganetespib can suppress the proliferation and growth of the HepG2 cell lines. The longer the acting time was, the higher the HepG2 cell proliferation inhibition was. There existed statistically significant (P<0.05) between the time-dose effect of a growing inhibition rate and HepG2 cell lines proliferation. Flow cytometry analysis showed that apoptosis rate of HepG2 cell line was ascending with the increasing concentration of Ganetespib. The apoptosis rate(16.3±1.22%) was significantly higher than the normal group (5.72±1.05%) when the the dose of drug was at an concentration of 30 nmol/L (P<0.05), while the 1000nmol/L Ganetespib group with apoptosis rate (56.6±1.83)% was higher than other low concentration groups (P＜0.05). ELISA showed the expression level of HSP90 was highest in 45’C group(compared with other groups, P<0.05).2. Ganetespib at 150mg/kg did not induce any influence in the appearance and micro-morphology of the heart, liver, spleen, lung and kidney at 5 hours, indicating that the inhibitor owned low toxic effect to the nude mice. Intravenous (IV) Ganetespib alone did not show obvious necrosis area 24h after treatment. Combination MWA-Ganetespib significantly increased coagulation size compared with MWA alone (9.±0.5mm vs.7.5±0.3mm P＜0.05). Combination MWA-Ganetespib decreased Hsp90 expression compared with MWA alone at 24 h (0.±0.07mm, 21.4±11.2%vs.0.25±0.13mm,30.3±10.7%, P<0.05). MWA-Ganetespib increased cleaved caspase-3 expression at 24 h (0.37±0.12mm,37.3±15.1% vs.0.25±0.18mm, 27.6±11.9%, P＜0.05).3. Differences in endpoint survival and tumor doubling time among the groups were highly significant (P<0.05). Endpoint survivals were 19.1±2.0 days for the control group,26.3±2.2 days for tumors treated with MWA alone,23.2±2.0 days for tumors treated with Ganetespib alone, and 33.2±1.9 days with combined MWA and Ganetespib. Tumor diameter doubling time in combined treatment group was (25.1 ±1.8 days) significantly longer than single microwave ablation group (17.5±1.6 days) and drug treatment group (15.4±2.3 days) alone (P< 0.05).Conclusions1. Heat shock protein 90 inhibitors Ganetespib can significantly inhibit the human liver cancer cell lines HepG2 proliferation, and the effect was time-dose. Tthe inhibitor can also induce HepG2 cell line apoptosis.2. Ganetespib owned low toxic effect to the nude mice. Suppression of HSP production using adjuvant Ganetespib can increase apoptosis and improve MWA ablation-induced tumour destruction.3. Ganetespib in combination with microwave ablation can reduce tumor growth rates and improves animal endpoint survival.