Two-thirds of patients with HCC are diagnosed in advanced stages receiving treatments as first- (Sorafenib and Lenvatinib) or second- (Regorafenib and Cabozantinib) line therapies2,3,4,5,6. However, drug resistance is widely observed and only a minor percentage of patients are effectively extending their survival after treatment21.
Sorafenib and Regorafenib exerted a more potent relative beneficial properties than Lenvatinib and Cabozantinib in reducing spheroid area (Fig. 1, Supplementary Table 1), cell proliferation (Fig. 2a, c, as well as Fig. 3), and promoting cell death (Fig. 2b, d, e, as well as Fig. 3b) in 3D and 2D cultured-differentiated HCC cells. However, a detailed analysis showed differences in drug response in 3D and 2D cell culture models. The reduction of spheroid area by Sorafenib and Regorafenib was similar in all cell lines cultured in spheroids (Fig. 1, Supplementary Table 1); however, the increase of caspase-3 activity, and at lower extent the reduction of BrdU incorporation, showed a significant decreasing order of effectiveness of the drugs in HepG2 ≥ Hep3B ≥ Huh7 cells cultured in monolayer (Fig. 3). Lenvatinib and Cabozantinib that reduced spheroid area in 3D cultured Huh7, but not in HepG2 and Hep3B (Fig. 1, Supplementary Table 1), appeared to reduce BrdU incorporation (Fig. 3a), and at low extent to increase caspase-3 activity (Fig. 3b), in all 2D cultured cell lines. These data might suggest that although a parallelism existed in drug response in 3D and 2D models, it can be feasible that the increased hypoxic environment in spheroids, which does not occur in cells cultured in monolayer, was inducing cell resistance to the antiproliferative and proapoptotic properties induced by the treatments. In fact, the enhanced expression of HIF-2alpha isoform causes a survival advantage in HepG2 cells cultured in spheroids22.
An increase in non-trypan blue-stained viable cells (Fig. 2a), trypan blue-stained non-viable cells (Fig. 2b), caspase-3 activity (Fig. 2d), and TUNEL-positive cells (Fig. 2e), and reduction in Ki67-positive cells (Fig. 2c) at day 10th were observed compared to day 15th in spheroids from control HepG2 cells. These changes suggested the existence of active cell proliferation at day 10th that progressed, but also coexisted with cell cycle arrest and apoptosis, at day 15th in spheroids. The presence of released autocrine factors or physical constraint might occur in our experimental conditions at day 15th after spheroid establishment. In addition, the strong reduction of Ki67-positive cells (Fig. 2c), and the increase of caspase-3 activity (Fig. 2d) and TUNEL-positive cells (Fig. 2e) induced by Sorafenib and Regorafenib at day 10th were associated with the reduction of non-trypan blue-stained viable cells (Fig. 2a), and the increase of trypan blue-stained non-viable cells (Fig. 2b) at day 15th in HepG2. This situation showed that the early activation of cell death and blockage of cell proliferation by Sorafenib and Regorafenib observed at day 10th had an impact in the number of viable and dead cells at day 15th in spheroids.
p53, a tumor suppressor gene involved in cell cycle control, DNA repair, apoptosis, and cell differentiation, is one of the most mutated genes (up to 67% according to the different studies) in HCC although mutation prevalence varies greatly depending on etiology23. The expression of p53 and other p53-related components of the gene family are transcriptionally regulating the expression of cell death receptors and apoptosis24,25. An association appears between p53 mutations, and the degree of cell dedifferentiation and survival in patients with HCC26,27. The experiments suggested that Sorafenib and Regorafenib compared with Lenvatininb and Cabozantinib were especially effective in HepG2 with wild-type 53, while a relative decreasing antiproliferative and proapoptotic properties were observed in 2D cultured Hep3B and Huh7 cell lines (Fig. 3a, b, respectively). This pattern of action is in agreement with our previous study that showed a more potent proapoptotic and antiproliferative effects of Sorafenib in HepG2 cells than Hep3b and Huh7 cells10. Sorafenib has also been shown to upregulated p53 and promotes p53-dependent apoptosis9,28. The lack of functional p53 in most HCC cells have been proposed as a possible mechanism for Sorafenib resistance28,29. The overexpression of p53 renders Hep3B more sensitive to Sorafenib, while p53 knockdown from HepG2 cells increased their resistance30.
The clinical trials have established recommended dose for Sorafenib (800 mg/24 h), Regorafenib (160 mg/24 h), Lenvatinib (<60 kg: 8 mg/24 h; ≥60 kg: 12 mg/24 h), and Cabozantinib (60 mg/24 h) in the treatment of patients in advanced stage of HCC. The dose–response analysis enclosed concentrations found in blood from advanced HCC-treated patients. Sorafenib (10 µM), and at lower extent Regorafenib (1 µM), exerted the strongest effects in cultured HepG2 cell line, while the response was lower in 2D cultured Huh7, SNU423, and SNU449 (Figs. 4 and 5). Although, Lenvatinib and Cabozantinib showed to exert moderate antiproliferative effects at their recommended dose in SNU423, and SNU423 and SNU449, respectively (Fig. 4c, d, respectively). However, our study also showed that Lenvatinib and Cabozanitib exerted increased activity at higher dose than those recommended (0.1 µM and 1 µM, respectively; Figs. 4 and 5).
The induction of cell death by Sorafenib and Regorafenib has been related to mitochondrial dysfunction in cultured hepatocytes and liver mitochondria fraction from rats31,32. We have observed that basal OCR and mitochondrial-dependent ATP generation of SNU423 and SNU449 were significantly lower than observed in HepG2, Huh7, and Hep3B (Table 1). In addition, HepG2 and Huh7, but not Hep3B cells, had higher maximal respiration capacity than SNU423 and SNU449 (Table 1). Hep3B and SNU449, which show increased number of mesenchymal phenotype markers than PLC/PRF/5 and Huh7, are associated with an increased expression of transforming growth factor β (TGF-β) and vimentin, as well as shunt glucose-6-phosphate to the pentose phosphate pathway (PPP), overall parallel with the reduction in E-cadherin expression and OCR33. The proapoptotic effectiveness of Sorafenib are associated with mitochondrial dysfunction and reduction of glycolysis in HepG2 cells19. In this study, the lack of effectiveness of low dose of Sorafenib (10 nM) compared with the recommended dose (10 µM) was associated with an increased mitochondrial function and glycolysis19. In concordance, Fiume et al.34 showed that the reduced decline of cell viability in SNU449 compared with that observed in PLC/PRF/5 by Sorafenib (4 and 8 µM) was associated with resistance to mitochondrial respiration downregulation. All this data might suggest that Lenvatinib and Cabozantinib, differently to Sorafenib and Regorafenib, have a relative higher antitumoral activity in cells with reduced mitochondrial respiration, increased glycolytic and PPP pathways that are associated with increased epithelial–mesenchymal transition (EMT) phenotype.
The resistance of HCC cells during Sorafenib administration has been related to the upregulation of survival cell signaling mediated through insulin growth factor receptor (IGFR)35,36, c-Met37, and FGFR36. c-Met phosphorylation and activation of mTOR are also related to resistance to Sorafenib in patient-derived HCC xenograft37. Tovar el al.36 have shown Sorafenib (5 µM) reduces cell viability in Hep3B (55%) and Huh6 (29%). Huh6 cell line, which express wild-type p53, shows increased number of markers and morphology features of undifferentiated cells than Hep3B cell line38. The resistance of Hep3B and Huh6 to Sorafenib is associated with FGFR36, and EGFR39, or FGFR36 upregulation, respectively. Linsitinib (5 µM), a dual TKI of IGF1R/IR, and the pan-FGFR inhibitor BGJ398, increased the effectiveness of Sorafenib in both resistant cell lines36. The increased effectiveness of Sorafenib and Regorafenib (Figs. 3–5), in comparison with Lenvatinib and Cabozantinib, was related to the downregulation of EGFR and c-Met protein expression (Fig. 6) in 2D cultured HepG2 cells.
Fernando et al.9 have shown that Sorafenib sensitizes HCC cells to the apoptotic activity of TGF-β through the intrinsic pathway and tumor necrosis factor-α (TNF-α)-dependent extrinsic pathway. The proapoptotic activity of Sorafenib is associated with downregulation of S-nitrosylation in cell death receptors in liver cancer cells40. In concordance with our study, untransformed hepatocytes did not respond to Sorafenib-induced cell death9. Sorafenib, Regorafenib, Lenvatinib, and Cabozantinib exerted a minor effect in cell proliferation and death in primary human hepatocytes (Figs. 4 and 5).
Different reports have suggested an association between EMT and chemoresistance in liver tumor cells. SNU449, HLF, and HLE liver cancer cell lines that express mesenchymal markers (CD44, vimentin, and snail) are refractory to Sorafenib treatment compared to HepG2, Hep3B, PLC/PRF/5-expressing epithelial markers (E-cadherin and CK-18)41,42. Similar conclusions were obtained by Van Zijl et al.43 using HCC cells derived from patients. In fact, factors turning back mesenchymal to epithelial phenotypes increase responsiveness of HCC cells to Sorafenib44. The expression of EMT markers appeared to be more relevant than upregulation of EGFR expression or downstream activation of ERK signaling for the sensitivity of tumor cells to EGFR inhibitors45. The EMT status also predicts HCC cell sensitivity to IGFR inhibitors in HCC cells46. A recent gene expression classification of HCC has identified a poor survival subclass termed S2 that express E-cadherin, c-myc, and FGFR3-4 protein expression47. Intriguingly, S2 gene signature that included Hep3B, HepG2, and HuH7, but not SNU423, cell lines that were highly susceptible to inhibition of cell proliferation by FGFR1-4 inhibitors47. In our conditions, Lenvatinib that targets FGFR3/4, showed lower antiproliferative and proapoptotic activities in HepG2, Hep3B, and HuH7 than SNU423 cell lines (Figs. 4–5).
In conclusion, the present study showed that although a parallelism existed in the effectiveness of drugs in well-differentiated cells cultured in 3D and 2D models, it might be that the hypoxic environment generated in spheroids would be responsible for the minor effectiveness of drugs in cells cultured in the 3D system in comparison with that observed in 2D cultured cells. The administration of regular used in vitro dose (10 µM) in 3D and 2D cultures, as well as the dose–response analysis in 2D cultures showed that Sorafenib and Regorafenib were increasingly effective reducing cell proliferation, and inducing apoptosis in well-differentiated and wild-type p53 HCC cells. Lenvatinib and Cabozantinib were more effective than Sorafenib and Regorafenib in moderately to poorly differentiated cells with mutated or lacking p53 HCC cells. The study also suggested that the highest effectiveness of Sorafenib and Regorafenib might be associated with high mitochondrial respiration, in comparison with Lenvatinib and Cabozantinib that appeared to be more active in cells with low basal OCR, mitochondrial-dependent ATP generation, and maximal respiration capacity.