AKT Phosphorylation Promotes Chemoresistance in Hepatocellular Carcinoma

Aim: This study was aimed to explore the effects of AKT phosphorylation in the chemoresistance and cell viability of hepatocellular carcinoma (HCC). Materials & methods: We developed two taxol-resisted hepatocellualr cancer cells: TAX Resis HepG2 and TAX Resis SMMC7721. Results: Phosphorylation of AKT on Thr 308 was highly expressed in taxol-resisted HepG2 and SMMC7721 cells. AKT phosphorylation manipulated by MK2206 and SC79 were correlated with cell viability. Downregulation of AKT phosphorylation promoted the apoptos is and suppressed the migration ability of taxol-resisted HepG2 and SMMC7721 cells, which also could be rescued by activate the AKT phosphorylation via SC79. Conclusion: AKT phosphorylation manipulated by MK2206 and SC79 were correlated with cell viability, migration and apoptosis in taxol-resisted HCC cells.


Introduction
Hepatocellular carcinoma (HCC) is the most common subtype of liver cancer and the fourth most frequent cause of death around the world 1 . In the last few decades, the treatment of HCC has been gradually improved, but its mortality rate remains high 2 . Only less than 30% of the HCC patients are suitable for radical resection or liver transplantation, and systemic chemotherapy is required for advanced HCC patients 3 . However, chemoresistance often develops 4 . The possible mechanisms of chemoresistance include disorder of the critical signal pathways, changes of the targets of anticancer drugs, and increased drug e ux 5 .
The activation of AKT kinase signaling pathway of human cancers is either indirectly through the activation of intersecting oncogenic pathways or directly through PI3 kinase, somatic mutation of PTEN, or AKT itself 6,7 . The AKT kinase, in turn, activates numerous downstream targets which could boost tumor survival, growth, and progression. As a result, most human tumors are believed to rely on varying degrees of AKT signaling to maintain their viability. Overexpression and activation of AKT are usually associated with resistance to radiotherapy or chemotherapy 8,9 . PTEN overexpression and PI3K inhibitors in PTEN-null cells have demonstrated the reversal of drug resistance 8,10 . The cytotoxicity of chemotherapeutic agents could be enhanced by dominant-negative mutants of AKT, which suggests that AKT plays a pivotal role in drug resistance 10,11 . Accordingly, small-molecule inhibitors of AKT suitable for clinical use have huge potential in the treatment of cancer 12,13 . More recently, accumulating evidences also have proved that AKT phosphorylation is highly expressed or overexpressed in chemoresistance tumor samples. However, the expression pattern and possible roles of AKT phosphorylation in HCC have not been investigated.
In this work, we conducted this study to investigate a role of AKT phosphorylation in the taxol-resisted hepatocellualr cancer cells: TAX Resis HepG2 and TAX Resis SMMC7721. We rstly found that AKT phosphorylation is overexpressed in taxol-resisted hepatocellualr cancer cells. We also observed that AKT phosphorylation regulates the growth, apoptosis and migration of taxol-resisted hepatocellualr cancer cells.

Cell lines
Human HCC cell line HepG2 and SMMC-7721 were obtained from the Cell Bank of the Institute of

Immuno uorescence
We placed round glass slides on the bottom of the wells of a 24-well plate and coated them with gelatin, then we seeded cells at a density of 1 × 10 4 cells per well. We incubated the slides initially with primary antibody against AKT phosphorylation [P-AKT (T308)] antibody (1:1000, 13038, Cell signaling technology, USA) at 4℃ in a humidi ed chamber for a night. Afterthat we added goat antimouse secondary antibody (labeled with FITC, 1:500 dilutions; Life Technologies) and incubated them for 1 h. Finally, we observed the slides under a uorescence microscope (Nikon) and captured the images using the NIS-Elements BR 4.20.00 software.

MTT assay
Taxol-resisted HepG2 and SMMC7721 hepatocellualr cancer cells were treated with MK-2206 (S1078, Selleck, USA) 9 µM for 72 hours (MK2206 group) and rescued with SC79 (HY18749, MCE, China) 1µg/Ml for 1 hour after removed MK2206 prior to analysis (MK2206 + rescued with SC79 group). The evaluation of cell proliferation was conducted using the MTT in accordance with manufacturer's instructions. In short, we added 10 µL of MTT solution to the culture medium, and incubated them for another 4 h. At 490 nm wavelength was the absorbance determined.

Cell apoptosis
Taxol-resisted HepG2 and SMMC7721 hepatocellualr cancer cells were processed with MK-2206 (9 µM) for 72 hours (MK2206 group) and rescued with SC79 (1µg/mL) for 1 hour after removed MK2206 prior to analysis (MK2206 + rescued with SC79 group). The measurements of apoptosis and necrosis in drug-

Cell migration assay
Fadu cells were grown to 50-70% con uence. We added the cells into the upper chamber of the insert (BD Bioscience, 8-µm pore size) for the migration assays. We incubated the cells in medium without serum, yet with 10% FBS in the lower chamber for chemo attractant in both assays. After a few hours of incubation, we wiped out the cells which did not migrate through the pores carefully with cotton wool. Finally, we stained the cells with 0.2% crystal violet and 20% methanol, and counted the imaged inserts.
Statistical analysis SPSS 21.0 software package (SPSS, Inc., Chicago, IL, USA) was applied for statistical calculations. The evaluation of the differences between two groups was conducted using the Student t-test. And one-way analysis of variance was applied to analyze the comparison of the means greater than or equal to three groups. Data were presented as the mean ± standard error of the mean (abbreviated as SEM). P < 0.05 was de ned as statistically signi cant.

Results
Establishment of taxol-resisted HepG2 and SMMC7721 hepatocellualr cancer cells We developed two taxol-resisted HCC cells: TAX Resis HepG2 and TAX Resis HepG2 SMMC7721. To test the taxol resistance ability of these cells, we cultured wide type and taxol-resisted HepG2 or SMMC7721 with indicated concentrations of Taxol (from 2 to 1280 nM). To determin the cell viability under taxol cultured environment, MTT assay were performed. As showed in Fig. 1A and B, the 50% inhibitory concentration (IC50) in HepG2 and SMMC7721 wide type cells were 77.30 ± 5.67 and 166.39 ± 5.32, while in HepG2 and SMMC7721 TAX Resis type ones were 405.46 ± 5.69 and 577.49 ± 504, respectively. The resistance index (RI) of Taxol-resisted cells of HepG2 and SMMC7721 were calculated based on the 50% inhibitory concentration (IC50), RI = 5.25 ± 0.07 for HepG2 and RI = 3.47 ± 0.03 for SMMC7721 (Fig. 1C).
The results indicated that these two TAX Resis types of HepG2 and SMMC7721 were successfully established.
Phosphorylation of AKT on Thr 308 was highly expressed in taxol-resisted HepG2 and SMMC7721 cells.
To investigate whether phosphorylation of AKT was highly expressed in TAX Resis types of HepG2 and SMMC7721 cells, western blot analysis were applied to analyze the phosphorylation levels of AKT on Thr 308 [p-AKT(T308)] and AKT (in total) ( Fig. 2A). We found that the average phosphorylation of AKT on Thr 308 [p-AKT (T308)] protein level in TAX Resis types of HepG2 and SMMC7721 cells was signi cantly higher than that in their wide types. Moreover, we performed immuno uorescence analysis of p-AKT (T308) in wide type and taxol-resisted HepG2 or SMMC7721 hepatocellualr cancer cells. The results showed that the expression of phosphorylation of AKT on Thr 308 [p-AKT (T308)] was signi cantly increased in TAX Resis types of HepG2 and SMMC7721 cells as compared with that in their wide types ( Figure. 2B). Together, these results suggested that phosphorylation of AKT on Thr 308 were highly expressed in taxol-resisted HepG2 and SMMC7721 cells.
AKT phosphorylation manipulated by MK2206 and SC79 were correlate with cell viability in Taxol-resisted HepG2 and SMMC7721 cells To investigate whether the expression level of AKT phosphorylation were correlated with cell viability, we manipulated the AKT phosphorylation level by the inhibitor (MK2206) and activator (SC79). Firstly, western blot analysis demonstrated that MK2206 decreased the level of AKT phosphorylation while SC79 activate the level of AKT phosphorylation in the dose response manner in HepG2 and SMMC7721 ( Fig. 3A and 3B). To measure the function of AKT phosphorylation downregulation and up regulation on the viability and proliferation ability of the Taxol-resisted HepG2 and SMMC7721 cells, MTT assays were performed. Taxol-resisted HepG2 and SMMC7721 hepatocellualr cancer cells were processed with MK-2206 (9 µM) for 72 hours (MK2206 group) and rescued with SC79 (1µg/mL) for 1 hour after removed MK2206 prior to analysis (MK2206 + rescued with SC79 group). MTT assay showed that AKT phosphorylation downregulation group (MK2206 group) were signi cantly inhibited the viability of the Taxol-resisted HepG2 and SMMC7721 cells compared with mock group and downregulation was rescued with SC79 group (Fig. 3C, *P < 0.05). These results indicated that the AKT phosphorylation manipulated by MK2206 and SC79 were correlate with cell viability in Taxol-resisted HepG2 and SMMC7721 cells.

Downregulation of AKT phosphorylation promoted the apoptosis of Taxol-resisted HepG2 and SMMC7721 cells
Since phosphorylation of AKT on Thr 308 was highly expressed in Taxol-resisted HepG2 and SMMC7721 cells, we would wonder whether high phosphorylation of AKT could reduce the apoptosis to resist the chemotherapy. To evaluated the apoptosis status, Taxol-resisted HepG2 and SMMC7721 hepatocellualr cancer cells were processed with MK-2206 (9 µM) for 72 hours (MK2206 group) and rescued with SC79 (1µg/mL) for 1 hour after removed MK2206 prior to analysis (MK2206 + rescued with SC79 group), and Annexin-V-PI assay were conducted. As showed in Fig. 4A, the upper right quadrant (Q2; Annexin-V+/PI+) implied apoptosis, AKT phosphorylation downregulation group (MK2206 group) were signi cantly promoted the apoptosis of the Taxol-resisted HepG2 and SMMC7721 cells compared with mock group and downregulation was rescued with SC79 group. The percentages of apoptosis parts were indicated in Fig. 4B. Furthermore, we have analyzed the expression level of two genes related with apoptosis (BCL-2 and Caspase3). BCL-2 were signi cantly reduced in AKT phosphorylation downregulation group (MK2206 group) compared with mock group and downregulation was rescued with SC79 group (Fig. 4C, **P < 0.01). While caspase3 were dramatically increased in AKT phosphorylation downregulation group (MK2206 group) compared with mock group and downregulation was rescued with SC79 group (Fig. 4D, **P < 0.01). Together, these results indicated that downregulation of AKT phosphorylation promoted the apoptosis of Taxol-resisted HepG2 and SMMC7721 cells, which also could be rescued by activate the AKT phosphorylation via SC79.
Regulation of AKT phosphorylation correlate with the migration potential of Taxol-resisted HepG2 and SMMC7721 cells To further explore the in uence of AKT phosphorylation on the migration potential of the Taxol-resisted HepG2 and SMMC7721 cells, transwell assays were used. The data showed that the number of migratory cells in the AKT phosphorylation downregulation group (MK2206 group) signi cantly reduced compared with mock group and downregulation was rescued with SC79 group (Fig. 5A and 5B, *P < 0.05). These data suggested that the downregulation of AKT phosphorylation suppressed the migration ability of Taxol-resisted HepG2 and SMMC7721 cells, which also could be rescued by activate the AKT phosphorylation via SC79.

Discussion
Although anticancer drugs like taxol, have been widely applied in the treatment of HCC, chemoresistance is still an important therapeutic di culty and its molecular mechanisms are poorly known. Our study demonstrated that PI3K/Akt pathway might be related to the chemoresistance in HCC. Our results demonstrated that MK-2206 and SC79 e caciously regulates Akt phosphorylation as well as inhibit the growth of taxol-resisted HepG2 and SMMC7721 cells. This nding is in accordance with previous studies that MK-2206 could e caciously inhibit the growth of different cancer cells, for instance, lung, colorectal, nasopharyngeal, and thyroid in vivo and in vitro [14][15][16][17] .
The activation of PI3K/Akt pathway plays a vital role in the biology of cancers, for instance, tumorigenesis, tumor metastasis, and the resistance to traditional chemotherapeutic agents 18 . The addition of paclitaxel was shown to increase Akt activity, which was in accordance with lower level of cell death 19 . Induction of Akt by cisplatin, as well, was in charge of the chemotherapeutic resistance observed 20 . Nevertheless, the exact mechanism by which Akt activation leads to chemoresistance is unknown. In this study, we demonstrated that AKT phosphorylation regulates chemoresistance in taxolresisted HepG2 and SMMC7721 cells.
Previous studies have demonstrated that the inhibition of PI3K/Akt could increase the induction of apoptosis in colon cancer cells incorporated with irinotecan and sensitize ovarian cancer cells to paclitaxel 21 . Molecule-targeted drugs that target other points of PI3K/Akt pathway, for instance LY294002, have been demonstrated to be able to restore the sensitivity of hepatocellular cancer cells to chemotherapy in vitro 22,23 . In the study, we found that AKT phosphorylation could be manipulated by two small molecule reagents MK2206 and SC79, which would further correlate with cell viability, migration and apoptosis.

Conclusion
AKT phosphorylation manipulated by MK2206 and SC79 were correlate with cell viability, migration and apoptosis in Taxol-resisted HepG2 and SMMC7721 cells, which may promote chemoresistance of HCC, and act as a valuable target for the treatment of HCC. This study provides important information for the identi cation and characterization of a new molecular target and a marker for HCC therapy.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
The data used to support the ndings of this study are available from the corresponding author upon request.

Competing interests
None.