Hypoxic HCC cells are more resistant to ATO than normoxic cells.
To evaluate the effect of oxygen concentration on the chemoresistance of HCC cells to ATO, 3 cell lines were respectively treated with increasing concentrations of ATO for 24-72 hours under normoxic and hypoxic conditions using the MTS assay. As shown in Figure 1a, both normoxic and hypoxic cells exhibited sensitivity to ATO in a dose- and time-dependent manner. Among these lines at different treatment points, HepG2 cells demonstrated more resistance to ATO, with the highest IC50 values, and Huh7 cells showed more sensitivity to ATO, with the lowest IC50 values (Figure 1b and Additional file Table 4). And the IC50 values were significantly higher under hypoxia than under normoxia at the same time-point, except for the IC50 for Huh7 cells at 72 hours.
To further verify that hypoxic HCC cells were more resistance to ATO, apoptosis assay were performed. Consistent with the above MTS results, hypoxic HCC cells showed less apoptosis than normoxic cells (Figure 1c). The above data indicate that hypoxic HCC cells are more resistant to ATO than normoxic ones.
HIF-1a levels are associated with the sensitivity of HCC cells to ATO.
We then performed qRT-PCR and western blot analyses to compare the difference of HIF-1a expression among 3 HCC lines and to determine whether HIF-1a levels affected the sensitivity of HCC to ATO. HepG2 cells expressed the highest endogenous levels of HIF-1a mRNA and protein under both normoxic and hypoxic conditions, whereas Huh7 showed the lowest levels of HIF-1a (Figure 1d). Taken together, these findings suggest that HCC cells with higher levels of HIF-1a are more resistant to ATO.
Hypoxic HCC cells upregulate HIF-1a protein expression post ATO-treatment in vitro.
As hypothesized, ATO could increase HIF-1a expression in hypoxic HCC cells, we analyzed the change of HIF-1a mRNA and protein expression levels in HCC cells treated with ATO under normoxic and hypoxic conditions. qRT-PCR analysis revealed that HIF-1a mRNA levels did not change in the 3 HCC lines in the absent or present of ATO (Figure 2a). However, western blot analysis indicated that HIF-1a protein expression showed a significant increase in all the three hypoxic HCC lines post-ATO exposure under hypoxic conditions, whereas there was no significant difference in normoxic cells, which may be associated with the rapid oxygen-dependent degradation of HIF-1a protein under normoxic conditions (Figure 2b). These results demonstrate that hypoxic HCC cells upregulate HIF-1a protein expression at the post-transcriptional level in response to ATO.
HIF-1a accumulation increases VEGF and P-glycoprotein synthesis in hypoxic HCC cells.
Mechanistically, accumulating stabilized HIF-1acan transcriptionally activate its target genes, such as multidrug resistance gene 1 (MDR1) and VEGF. MDR1 encodes for P-glycoprotein, the overexpression of which decreases the concentration of intracellular drugs and is one of the most common reason for chemotherapeutic resistance, such as ATO [15, 16], and VEGF plays a key role in tumor angiogenesis and proliferation [28]. As shown in Figure 2a, ATO-induced HIF-1aaccumulation stimulated a 2.2–2.8-fold and 4.2–5.8-fold increase of MDR1 and VEGF mRNA expression in hypoxic HCC cells compared with normoxic cells, whereas neither of them showed an increase in normoxic cells. Furthermore, MDR1 and VEGF mRNA levels were markedly higher post-ATO exposure than hypoxia-alone. Western blot analysis further confirmed the increased levels of P-glycoprotein and VEGF expression in the present of ATO under hypoxia (Figure 2b).
Both HIF-1a silencing and inhibition can enhance the sensitivity to ATO by downregulating P-glycoprotein and VEGF expression
To determine the role of HIF-1a upregulation in the acquired resistance to ATO, we tested whether inhibition of HIF-1a overexpression in hypoxic HCC cells can restore their sensitivity to ATO. Among the three duplexes of siRNA, siHIF-1a_3 at 50 nM concentration was confirmed to result in the highest inhibition of HIF-1a expression in hypoxic HepG2 cells, accompanied with complete P-glycoprotein and VEGF expression suppression (Additional file Figure S1a and Figure 3a). This siRNA was used in subsequent experiments to silence HIF-1amRNA. Cell viability assay indicated that HIF-1a silencing could significantly enhance the chemosensitivity of the hypoxic HCC cells to ATO (Additional file Figure S1b).
Next, we conducted YC-1 to facilitate HIF-1a protein degradation. Western blot analysis indicated that YC-1 could inhibit ATO-induced HIF-1a accumulation dose dependently and effectively suppress the expression of HIF-1a, P-glycoprotein, and VEGF at 20 μM (Additional file Figure S2a and Figure 3b). CDI studies showed that, in the presence of 20 μM ATO under hypoxic conditions, YC-1 enhanced the cell proliferative inhibition of ATO in the three HCC lines, and all CDIs values were less than 1, suggesting the synergistically inhibitory effect due to their combination (Additional file Figure S2b). On the basis of these results, the optimal concentration of YC-1 on inhibiting HIF-1a accumulation was 20 μM, which was used in the following combination assays.
To investigate HIF-1a inhibition on HCC cell apoptosis, the apoptosis assay was performed in HepG2 cells. Confirmed HIF-1a inhibition by western blot, YC-1 and siHIF-1a combined with ATO could significantly increase apoptosis compared with ATO-alone under hypoxia (Figure 3c-d).
Taken together, these findings strongly suggest that HIF-1a upregulation in hypoxic HCC cells post-ATO treatment is involved in acquired chemoresistance to ATO in vitro, and targeting HIF-1a could reverse the ATO-induced chemoresistance of HCC cells.
ATO-induced HIF-1a accumulation increases VEGF secretion and stimulates angiogenesis in vitro.
As shown in Figure 2c, secretory VEGF levels in the culture supernatants of HepG2 cells examined by ELISA were markedly increased, further confirming the results obtained qRT-PCR and western blot analysis. With 40 μM ATO treatment, the surviving hypoxic HCC cells could maintain a relative high level of VEGF secretion. Importantly, the culture supernatant prepared from 10 μM ATO-treated cells under hypoxic conditions induced a significant pro-angiogenesis compared with the control (Figure 2d). Consistent with these findings, the angiogenesis effect of the supernatant was inhibited by blocking HIF-1a expression using HIF-1a siRNA or inhibitor (Figure 3e).
ATO-induced HIF-1a upregulation attenuates the sensitivity of HCC tumors to ATO and contributes to the acquired ATO-resistance in vivo.
To confirm an alteration of HIF-1a expression and its role on ATO-resistance in vivo, we administered nu/nu mice bearing subcutaneous HepG2 tumors with YC-1, ATO or both in combination over a 3-week period. As shown in Figure 4a, YC-1 did not significantly increase ATO toxicity on weight loss in mice. Although tumors treated with ATO were decreased to 60.9% in volume as compared with the control tumors (1905 ± 172 mm3 vs. 1160 ± 171 mm3, p < 0.001), ATO and YC-1 exerted a more significant antitumor effect than ATO-alone on subcutaneous HCC tumors in mice (1160 ± 171 mm3 vs. 327 ± 78 mm3, p < 0.001) (Figure 4b). Tumor weight further confirmed the synergistic inhibitory effect of YC-1 combined with ATO (Figure 4c).
We further analyzed tumors by H&E staining and immunohistochemistry and western blot analyses. Tumors treated with ATO showed effective tumor-growth inhibition, foci of necrosis, and a decrease of microvessel density (MVD), whereas there was an significant increase of HIF-1a, P-glycoprotein, and VEGF expression in ATO-therapy tumors compared with the control tested by western blotting (Figure 5a-c). These findings reveal that ATO at 4mg/kg/day is effective on antitumor growth and could simultaneously induce upregulation of HIF-1a, VEGF, and P-glycoprotein, as hypothesized.
However, ATO in combination with YC-1 produced enhanced antitumor effects with greater necrosis in tumors than ATO alone, which may result from, at least partially, lower MVD in the combinational tumors (Figure 5a-b). In line with our observations in vitro, the increased protein levels of P-glycoprotein and VEGF were remarkably suppressed with the inhibition of ATO-induced HIF-1aoverexpression in tissue lysates prepared from tumours treated with YC-1 and the combination (Figure 5c). In addition, HIF-1a inhibition could also augment the antitumor effects of ATO via an increase of apoptosis and a decrease of proliferation in HCC tumors (Figure 5a-b). Taken together, these results confirmed that ATO-induced HIF-1a upregulation attenuates the sensitivity of HCC to ATO and contributes to the molecular mechanism of acquired resistance in vivo.