Combination chemotherapy with oxaliplatin and saracatinib exhibited antagonistic effects
The efficacy of combinations between oxaliplatin and anti-cancer molecular targeting drugs was screened. Strangely, the combined chemotherapy with oxaliplatin and saracatinib induced significantly antagonistic effects (Figure 1A). The HCC cell lines MHCC97L and Hep3B exhibited significant anti-tumor effects with lower proliferation rates than wild-type cells following treatment with oxaliplatin or saracatinib individually; however, combination treatment with the two drugs resulted in a higher proliferation rate, indicating impaired antitumor efficacy with combined treatment (Figure 1B). To confirm this phenomenon, plate colony formation assays were performed. Compared to untreated cells, colony diameters were significantly decreased following treatment with oxaliplatin (257.51±55.60 μm vs. 705.16±170.81 μm; p=0.041) or saracatinib (287.57±71.36 μm vs. 705.16±170.81 μm; p=0.0025) individually. Combination therapy, on the other hand, resulted in larger colony diameters than oxaliplatin (455.16±86.12 μm vs. 257.51±55.60 μm; p=0.0086) or saracatinib (455.16±86.1 μm vs. 287.25±71.36 μm; p=0.0245) treatment alone (Figure 1C). Next, we analyzed the cell cycle distributions to further evaluate the observed changes in cell proliferation. The percentages of cells in S phase decreased following treatment with either oxaliplatin (22.321±0.67% vs. 29.48±1.06%; p＜0.0001) or saracatinib (23.59±1.76% vs. 29.48±1.06%; p=0.003). Combination therapy with oxaliplatin and saracatinib resulted in significantly increased percentages of cells in S phase compared to treatment with oxaliplatin (26.55±0.39% vs. 22.321±0.67%; p＜0.0001) or saracatinib (26.55±0.39% vs. 23.59±1.76%; p=0.0026) individually (Figure 1D). Furthermore, the tumor weight of the subcutaneous xenografts were larger in the combination treatment group than oxaliplatin (3.06±0.16 g vs. 1.51±0.39 g; p=0.0005) or saracatinib (3.06±0.16 g vs. 1.97±0.32 μm; p=0.0008) treatment alone (Figure 1E).
We next revalidated the effects of the combined therapy on the response of HCC cells. MHCC97L (130.6±16.62 μmol/L vs. 20.85±4.86 μmol/L; p=0.0063) and Hep3B (28.67±5.59 μmol/L vs. 5.29±1.29 μmol/L; p=0.0247) cells exhibited significantly increased IC50 values in response to oxaliplatin when treated with saracatinib. Interestingly, however, MHCC97L (0.79±0.11 μmol/L vs. 4.81±0.57 μmol/L; p=0.0056) and Hep3B (2.05±0.32 μmol/L vs. 2.62±0.47 μmol/L; p=0.0631) cells exhibited reduced IC50 values in response to saracatinib when treated with oxaliplatin as well (Figure 2A). These findings suggest that saracatinib treatment in combination with oxaliplatin increases oxaliplatin resistance in HCC, and it was the treatment with saracatinib caused oxaliplatin resistance. Additionally, we investigated the effects of the two chemotherapy drugs on protein expression in the two HCC cell lines MHCC97L and Hep3B. Oxaliplatin treatment led to the downregulation of PCNA and the occurrence of EMT, which was associated with the upregulation of vimentin and the downregulation of E-cadherin. Conversely, saracatinib not only downregulated PCNA expression but also reversed the EMT. MHCC97L and Hep3B treated with both oxaliplatin and saracatinib exhibited partial upregulation of PCNA and reversion of EMT (Figure 2B). These findings suggest that saracatinib treatment in combination with oxaliplatin reduces the antitumor efficacy of these drugs on HCC cells, but reverses the negative effect of EMT induced by oxaliplatin.
Sequential chemotherapy reduced the antitumor efficacy of oxaliplatin on saracatinib-resistant HCC
In order to simulate the clinical sequential chemotherapy, HCC cell lines were treated continually with oxaliplatin to generate oxaliplatin-resistant cell lines (MHCC97L-Oxa and Hep3B-Oxa) that exhibited decreased intercellular adhesion and spindle-shaped cell morphology (Figure 3A). Compared to wild-type HCC cells, MHCC97L-Oxa (66.67±9.01 μmol/L vs. 31.67±4.04 μmol/L; p=0.0254; Figure 3B, a) and Hep3B-Oxa (19.21±2.69 μmol/L vs. 5.45±1.23 μmol/L; p=0.0212; Figure 3B, c) exhibited increased oxaliplatin IC50 values. The oxaliplatin-resistant HCC cell lines were next treated with increasing concentrations of saracatinib, resulting in a decrease of IC50 values in MHCC97L-Oxa (1.23±0.31 μmol/L vs. 4.30±0.97 μmol/L; p=0.0141; Figure 3C, a) and Hep3B-Oxa (1.14±0.11 μmol/L vs. 2.62± 0.47 μmol/L; p=0.0333; Figure 3C, b) compared to the parental wild-type HCC cells, still exhibiting more sensitive to saracatinib.
Saracatinib-resistant MHCC97L and Hep3B cells (MHCC97L-Src and Hep3B-Src) were generated similarly via continuous treatment with saracatinib, and these lines exhibited enhanced intercellular adhesion and appeared as agglomerated cell clumps (Figure 3A). Compared to wild-type cells, MHCC97L-Src (40.07±2.88 μmol/L vs. 4.81±0.57 μmol/L; p=0.0024; Figure 3B, b) and Hep3B-Src (38.36±3.17 μmol/L vs. 2.62±0.47 μmol/L; p=0.009; Figure 3B, d) exhibited increased saracatinib IC50 values. The saracatinib-resistant HCC cell lines were then treated with oxaliplatin at increasing concentrations, yielding significantly enhanced resistance to oxaliplatin with increased IC50 in both MHCC97L-Src (108.71±11.24 μmol/L vs. 20.85±4.86 μmol/L; p=0.0092; Figure 3D, a) and Hep3B-Src (27.01±4.59 μmol/L vs. 5.29±1.29 μmol/L; p=0.0106; Figure 3D, b) compared to wild-type cells.
Additionally, we investigated protein expression in oxaliplatin- and saracatinib-resistant cell lines. MHCC97L-Oxa and Hep3B-Oxa exhibited downregulation of PCNA and E-cadherin and upregulation of vimentin and OCT4, while MHCC97L-Src and Hep3B-Src exhibited downregulation of PCNA, upregulation OCT4, and reversion of EMT compared to the parental cell lines (Figure 3E). Together, these results indicate that sequential chemotherapy reduced the antitumor efficacy of oxaliplatin on saracatinib-resistant HCC.
ABCG1 upregulation and Wnt signaling pathway activation are integral mechanisms involved in the antagonism between saracatinib and oxaliplatin in HCC
The expression of 20,030 genes was compared between wild-type MHCC97L and MHCC97L-Src cells in three independent experiments (Figure 4A). Gene expression profiles for 1172 genes exhibited differences (p<0.05) between MHCC97L and MHCC97L-Src, implicating these genes in saracatinib resistance. Furthermore, analysis of these associated “resistance” genes revealed 526 upregulated and 645 downregulated genes. The expression of these 20,300 genes in wild-type MHCC97L and MHCC97L-Oxa was also compared (Figure 4B). Expression profiles for 720 genes exhibited differences (p<0.05) between MHCC97L and MHCC97L-Oxa, implicating these genes in oxaliplatin resistance. Of these, 455 were upregulated, and 265 were downregulated in the two drug-resistant cell lines.
A total of 458 altered genes overlapped between the two drug-resistant cell lines, and all of these were closely related to cell division, growth, angiogenesis, adhesion, and metabolic processes (Figure 4C). KEGG pathway analysis revealed that 20 of the altered genes were related to drug resistance: ABCG1, ATM, BBC3, BIK, BIRC3, CDKN1A, DLL4, ERBB3, FGF2, FOS, GPER1, IL6, JAG1, MMP2, NRG2, PDGFRB, PIK3CA, SHC4, TOP2A, TOP2B, and VEGFA. Another 16 genes were related to Wnt signaling: ROCK2, TCF7L1, WNT6, WNT5A, NKD2, FZD3, FZD8, DKK1, WNT5B, NFATC4, PLCB2, SERPINF1, AXIN2, NFATC1, PLCB4, and RAC2. Immunoblotting confirmed the upregulation of ABCG1 and Wnt-associated proteins, including FZD8, DKK1, Axin2, and WNT6 (Figure 4D, E, F and G). Immunohisochemotherapy verified that the expression of ABCG1 was significantly upregulated after the treatment with oxaliplatin (16.25±4.03 vs. 7.50±3.42 μmol/L; p=0.0162) or saracatinib (20.50±4.51 vs. 7.50±3.42 μmol/L; p=0.0037) in subcutaneous xenografts tissues. And the combination treatment exhibited higher expression of ABCG1 than oxaliplatin single use (30.50±5.01 vs. 16.25±4.03; p=0.0044; Figure 4H). Therefore, we speculate that ABCG1 upregulation and Wnt signaling pathway activation are integral mechanisms involved in the antagonism between saracatinib and oxaliplatin in HCC.
Interference with ABCG1 expression or inhibition of Wnt signaling resulted in reversal of the saracatinib-induced oxaliplatin resistance in HCC
Immunoblotting verified that the expression of ABCG1 was significantly downregulated by Wnt/β-catenin signaling pathway inhibition with KYA1797K in wild-type HCC cell lines, MHCC97L-Src, and Hep3B-Src (Figure 5A). Following ABCG1 downregulation and, the key cell membrane receptors for Wnt signaling LRP6 and p-LRP6 were not significantly altered; however, β-catenin was slightly downregulated, and the expression of PCNA was significantly decreased. ABCG1 restoration could reverse this alteration in protein levels (Figure 5B).
Next, we confirmed the role of ABCG1 and Wnt signaling in oxaliplatin resistance. ABCG1 was silenced using specific siRNA in MHCC97L cells, resulting in a decreased IC50 to oxaliplatin compared to mock-treated MHCC97L cells (MHCC97L-Mock; 8.41±2.09 μmol/L vs. 25.59 ± 5.82 μmol/L; p=0.0085; Figure 5C, a). Following silencing of ABCG1 using siRNA in saracatinib-resistant MHCC97L (MHCC97L-Src-ABCG1-Sh1), we observed decreased resistance to oxaliplatin (40.43±8.12 μmol/L vs. 103.71±8.74 μmol/L; p=0.0008; Figure 5C, b) compared to mock-treated cells. Furthermore, silencing of ABCG1 combined with saracatinib in MHCC97L, there was no significantly increased resistance to oxaliplatin (11.84 ± 2.11 vs. 9.73±1.26 μmol/L; p=0.0874; Figure 5C, c). Additionally, HCC cells treated with the Wnt signaling inhibitor KYA1797K exhibited decreased resistance to oxaliplatin with reduced IC50 values (11.07±2.02μmol/L vs. 31.67±4.04μmol/L; p=0.0082; Figure 5D, a). Treatment of MHCC97L-Src cells with KYA1797K resulted in decreased resistance to oxaliplatin (40.83±8.12 μmol/L vs. 108.71±11.24 μmol/L; p=0.0008; Figure 5D, b). KYA1797K combined with saracatinib in MHCC97L, there was no significantly increased resistance to oxaliplatin (11.51 ± 1.44 vs. 15.73±2.95 μmol/L; p=0.0920; Figure 5D, c).
Furthermore, it was confirmed once again that the tumor weight of the subcutaneous xenografts was larger in combination treatment group using oxaliplatin and saracatinib than oxaliplatin single use (1.82±0.30 g vs. 1.26±0.23 g; p=0.0050; Figure 5E). And the subcutaneous xenografts was smaller in the combination treatment group using oxaliplatin, saracatinib, and ABCG1 siRNA local injection (0.87±0.24 g vs. 1.82±0.30 g; p=0.0001; Figure 5E) than combination group only using oxaliplatin and saracatinib. Together, these findings suggest that ABCG1 and Wnt signaling contribute to oxaliplatin resistance in saracatinib-treated HCC cells. And interference with ABCG1 expression or inhibition of Wnt signaling resulted in reversal of the saracatinib-induced oxaliplatin resistance in HCC