Effects Of STAT3Gene Silencing And Rapamycin On Apoptosis In Hepatocarcinoma Cells

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide andHCC-associated annual mortality ranks the third among all tumors. Its incidence, however, isstill on the rise, and is predicted to plateau between2015and2020. Despite the advancementof different therapeutic approaches, such as surgical resection, liver transplantation,interventional therapy, and percutaneous ablation, improvements in patients' outcomes arestill quite limited. Therefore, there is an urgent need to develop novel therapies thateffectively target the pathological alterations underlying HCC.In complex biological systems, cells communicate with their microenvrionment throughintertwined regulatory signaling networks. In response to exogenous signals, cellsdifferentiate, proliferate, or undergo cell death/apoptosis in order to maintain constantnumbers and support organ functions. Biological homeostasis in cell growth is achieved andfine tuned by activating, as well as inhibitory signaling pathways. The constitutivefunctioning of the former and/or the diminished performance/loss of the latter causessustained signal transduction leading to uncontrolled cell proliferation, resistance to apoptosisand subsequently, malignant growth. Accumulating evidence demonstrates that abnormalitiesin many signaling pathways are associated with the pathogenesis of HCC.Mammalian target of rapamycin (mTOR) is a key molecule in the PI3K/Akt/mTORsignaling pathway that, through the activation of various downstream targets, criticallyregulates cell proliferation, cell cycle progression, gene transcription, protein synthesis, andcellular apoptosis. Previous studies indicate that mTOR is frequently (67%,37/55)overexpressed in human HCCs, as detected by real-time reverse transcriptase-polymerase chain reaction (RT-PCR), and consistently, the mTOR pathway is activated in40-50%of HCCpatients.The signal transducer and activator, STAT3regulates the expression of target genesinvolved in cell-cycle progression and apoptosis, and promotes cellular transformation as wellas abnormal cell proliferation. The deregulation and constitutive activation of STAT3arefrequently observed in a large number of primary tumors and cancer-derived cells includinghematologic malignancies, breast cancer, prostate cancer, and HCC.Studies have shown that mTOR phosphorylates STAT3at Ser727, which is required forthe latter to maximally activate the transcription of target genes. In light of the crosstalkbetween mTOR and STAT3, and their frequent activations in HCC, we wanted to explore thefunctional consequences of targeting these two molecules on cellular apoptosis. Toaccomplish this, pGC-siRNA-STAT3plasmid were constructed, which was transfected intoBEL-7402cells using Lipofectamine2000, silencing the expression of STAT3to inhibitthe JAK/STAT3signaling pathway. Meanwhile we inhibited the phosphorylation of mTORusing rapamycin (109nmol/L) to inhibit the PKB/Akt/mTOR signaling pathway.Following treatment, Annexin V/propidium iodide staining followed by flow cytometryand Hoechst33258immunofluorescence staining was used to examine cellular apoptosis. JC-1staining was used to monitor depolarization of mitochondrial membrane (ΔΨm). Furthermore,the expression of activated caspase3protein was analyzed by Western blotting.In this study, our results showed that, p-mTOR level was significantly reduced in Rapacells, as compared to in control cells (P <0.05), while the level of total mTOR was notdramatically affected (P>0.05). Rapamycin significantly inhibits the phosphorylation ofmTOR in Bel-7402cells.To reduce the expression of STAT3, we transfected Bel-7402cells with pGC-siRNA-STAT3. As a control, pGC-siRNA-scramble plasmid was transfected. Compared to thenon-transfected cells, siRNA-scramble cells showed no reduction of STAT3level, while siRNA-STAT3cells exhibited approximately50%reduction in STAT3level (P<0.05).STAT3siRNA specifically knocks down STAT3protein level in Bel-7402cells.The percentage of apoptotic cells, as represented by dual PI and Annexin V positivity,ranged from9.22±0.38%in non-treated BEL-7402cells to16.47±1.04%in siCtrl,42.73±0.88%in siRNA-STAT3,43.03±0.46%in rapamycin-treated,45.44±0.59%insiRNA-scramble+Rap, and60.22±0.87%in siRNA-STAT3+Rap cells. Targeting eitherSTAT3with siRNA or mTOR with rapamycin significantly promoted apoptosis, as comparedto non-treated or siRNA-scramble-transfected cells (P<0.05). This pro-apoptotic effect wasfurther enhanced when both molecules were targeted (P<0.05) Targeting STAT3and/ormTOR promotes cellular apoptosis.Consistent with FCM analysis, BEL-7402cells also presented typical apoptoticmorphology following treatment with siRNA-STAT3, rapamycin, or both. These treatmentsnot only reduced the number of cells remaining attached to the plate but also led tocharacteristic changes of chromatin condensation and nuclear fragmentation, with the mostdramatic changes observed in cells treated with both siRNA-STAT3and rapamycin.Targeting STAT3and/or mTOR leads to mitochondrial depolarization in BEL-7402cells(27.28±1.82%, P<0.05, as compared to all other groups). Besides mitochondrialdepolarization, the expression of cleaved/activated caspase3in BEL-7402cells followingdifferent treatments was analyzed by Western blotting. Targeting STAT3and/or mTORincreases cleaved caspase3levels in BEL-7402cells.Hepatocarcinogenesis is strongly linked to deregulation of major signaling pathwayssuch as PI3K/Akt/mTOR and JAK/STAT3. In this study, we explored the potential oftargeting these two signaling pathways and their effects on regulating cellular apoptosis.Apoptosis, or programmed cell death, is a complicated, genetically determined processinvolved in the development and maintenance of homeostasis in multicellular organisms. Thecapability of evading apoptosis is critical for the development and sustained growth of many, perhaps all, cancers, and the induction of apoptosis has now been considered as an importantapproach for cancer therapy. In this study, we showed that targeting either STAT3or mTORsignificantly induced apoptosis in BEL-7402cells; in addition, the combined treatmentdisplayed further enhancement in apoptosis, as demonstrated by both FCM and Hoechst33258staining.It is known that apoptosis can be triggered in a cell through either the extrinsic/deathreceptor-mediated or intrinsic/mitochondria-mediated pathway. Thus, we further characterizedthe nature of apoptosis induced by targeting STAT3and/or mTOR. We found that followingtreatment with either siRNA-STAT3or rapamycin, there was significant decrease in themitochondrial membrane potential, as indicated by the decrease in JC-1dye aggregates (red)and increase in its monomers (green). The mitochondrial depolarization became evendramatic with combined treatments, suggesting that both STAT3and mTOR are essential formaintaining mitochondrial integrity. The ΔΨm depolarization triggers the release ofcytochrome c that activates caspases in the cytosol and subsequently initiates the apoptoticcascade. Consistently, Western blot showed that the expression of cleaved/activated caspase3by treatment with both siRNA-STAT3and rapamycin is up-regulated, significantly higherthan that in other groups.Although the combined treatments with siSTAT3and rapamycin increased apoptosis,depolarized the mitrochondrial membrane, and up-regulated cleaved caspase3, to a greaterextent than either treatment alone, we did not observe an additive effect. We made theconjecture that the cause involves two factors. On one hand, the improved pro-apoptoticactivity of combined treatments suggests that these two signaling pathways do not convergeon the exact same targets to regulate apoptosis. On the other hand, the short of additive effectsimplies that they do not work mutually exclusively in modulating apoptosis. This is consistentwith a previous observation that mTOR directly phosphorylates STAT3, which is required forthe optimal activity of the latter to regulate downstream targets. In conclusion, the present work demonstrates that combined treatments using rapamycinand STAT3gene silencing more potently promotes apoptosis in BEL-7402cells, as comparedto targeting either molecule alone. This study lays the groundwork for future development ofmulti-target drugs in cancer therapy.