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 treated with increasing concentrations of ATO for 24-72 hours under normoxic and hypoxic conditions and analysed 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 cell lines, HepG2 cells demonstrated higher resistance to ATO, with the highest IC50 values, and Huh7 cells showed more sensitivity to ATO, with the lowest IC50 values at each time point (Figure 1b and Additional file Table 4). The IC50 values for the cell lines were significantly higher under hypoxic conditions than under normoxic conditions at the same time-point, except for the IC50 for Huh7 cells at 72 hours.
To further verify that hypoxic HCC cells were more resistant to ATO, apoptosis assays were performed. Consistent with the above MTS results, hypoxic HepG2 cells showed lower apoptosis rates than normoxic cells (Figure 1c). The above data indicate that hypoxic HCC cells are more resistant to ATO than normoxic HCC cells.
HIF-1a levels are associated with the ATO sensitivity of HCC cells.
We then performed qRT-PCR and western blot analyses to compare the difference in HIF-1a expression among the 3 HCC lines and to determine whether HIF-1a levels affected the sensitivity of HCC to ATO. As shown in Figure 1d-e, HepG2 expressed the highest endogenous levels of HIF-1a mRNA and protein under both normoxic and hypoxic conditions, whereas Huh7 expressed the lowest levels of HIF-1a. Taken together with above, these findings suggest that HCC cells with higher levels of HIF-1a are more resistant to ATO. These results are in agreement with a previous study by Tung et al.  and extend these findings.
Hypoxic HCC cells upregulate HIF-1a protein expression post-ATO treatment in vitro.
We hypothesized that ATO could increase HIF-1a expression in hypoxic HCC cells, we analyzed the change in 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 absence or presence of ATO (Figure 2a). However, western blot analysis indicated that HIF-1a protein showed more expression in all the three hypoxic HCC lines post-ATO exposure under hypoxic conditions, whereas there was no upregulation in normoxic cells; this finding may be associated with the rapid oxygen-dependent degradation of HIF-1a protein under normoxic conditions (Figure 2b) [6, 25]. 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-1a can transcriptionally activate its target genes, such as multidrug resistance gene 1 (MDR1) and VEGF. MDR1 encodes P-glycoprotein, the overexpression of which decreases the concentration of intracellular drugs and is one of the most common reasons for chemotherapeutic resistance, such as ATO resistance [15, 16], and VEGF plays a key role in tumor angiogenesis and proliferation . As shown in Figure 2a, ATO-induced HIF-1aaccumulation stimulated a 2.2–2.8-fold and 4.2–5.8-fold increase in MDR1 and VEGF mRNA expression, respectively, 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 with ATO exposure than with hypoxia alone. Western blot analysis further confirmed the increased levels of P-glycoprotein and VEGF expression in the presence of ATO under hypoxic conditions (Figure 2b).
Increased VEGF secretion by HIF-1a accumulation 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 maintained a relatively high level of VEGF secretion. Importantly, compared with the control supernatant, the culture supernatant obtained from 10 μM ATO-treated cells under hypoxic conditions induced a significant pro-angiogenic compared with the control (Figure 2d).
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 by complete P-glycoprotein and VEGF expression suppression (Additional file Figure S1a and Figure 3a). This siRNA was used in subsequent experiments to silence HIF-1a mRNA. Cell viability assays indicated that HIF-1a silencing could significantly enhance the chemosensitivity of hypoxic HCC cells to ATO (Additional file Figure S1b).
Next, we treated cells with 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 CDI values were less than 1, suggesting the synergistic inhibitory effect due to their combination (Additional file Figure S2b). On the basis of these results, the optimal concentration of YC-1 for inhibiting HIF-1a accumulation was 20 μM, which was used in the following combination assays.
To investigate HIF-1a inhibition of HCC cell apoptosis, an apoptosis assay was performed in HepG2 cells. Confirming HIF-1a inhibition by western blotting, YC-1 or siHIF-1a combined to that with ATO could significantly increase apoptosis of HepG2 cells compared with ATO alone under hypoxic conditions (Figure 3c-d). Furthermore, the angiogenic effect of the supernatant of HepG2 cells was significantly inhibited by blocking HIF-1a expression using HIF-1a siRNA or inhibitor (Figure 3e).
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 that targeting HIF-1a could reverse the ATO-induced chemoresistance of HCC cells.
ATO-induced HIF-1a upregulation attenuates the sensitivity of HCC tumors to ATO and contributes to acquired ATO resistance in vivo.
To confirm an alteration of HIF-1a expression and its role in 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 or weight loss in mice. Although tumors treated with ATO were decreased to 60.9% in volume 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 analysed tumors by H&E staining and immunohistochemistry and western blot analyses. Tumors treated with ATO showed effective tumor growth inhibition, foci of necrosis, and decreased microvessel density (MVD), whereas there was a significant increase in HIF-1a, P-glycoprotein, and VEGF expression in ATO treated tumors compared with control, as tested by western blotting (Figure 5a-c). These findings reveal that ATO at 4 mg/kg/day is effective for inhibiting tumor 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 partially result from, lower MVD in the combination-treated 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-1a overexpression in tissue lysates prepared from tumors treated with YC-1 and the combination (Figure 5c). In addition, HIF-1a inhibition could also augment the antitumor effects of ATO via promoting the apoptosis and inhibiting the proliferation of 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.