الفهرس 
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Abstract
Hepatocellular carcinoma (HCC) is categorized as the sixth and fourth common cancer in worldwide and Egypt, respectively. In Egypt, HCC constitutes a significant public health due to the increasing incidence and complications of hepatitis C virus (HCV), which is the most important risk factor in developing liver cancer including HCC.
Surgical resection, liver transplantation, and ablation are different lines of therapy that offer a high rate of complete excision of disease and, hence, the potential for cure. However, the disease frequently relapses or is even diagnosed at an advanced stage when curative treatments are no longer accessible. Furthermore, no adjuvant therapy has been demonstrated to improve recurrence-free survival after curative treatments. Sorafenib, a tyrosine kinase inhibitor, is considered the standard of care for patients with advanced disease. However, it is poorly tolerated and has limited benefits due to frequent drug resistance. Therefore, there is an urgent need to develop alternative systemic therapies to improve patients’ outcomes.
Immune checkpoint inhibitors (ICIs) are thought to be one of the most promising cancer treatment strategies. Among these ICIs is those targeting (PD-1/PD-L1) axis which is overexpressed on HCC cells and is supposed to play a crucial role for cancer cells to escape from immune surveillance. The PD-1/PD-L1 axis blockade can reactivate the dysfunctional T cells and hence has been considered the key strategy to counteract the immune resistance of cancer. PD-L1 is broadly upregulated in many types of cancer cells Interestingly, durable responses have been recently reported in HCC with nivolumab, an anti-PD-1 monoclonal antibody. However, the efficacy of monotherapy for PD-1/PD-L1 blockade was rarely more than 40%.
PD-L1 inhibitor (BMS-1) used in the current study is a small molecule inhibitor which may exert advantages over monoclonal antibodies, having fewer side effects, better tumor penetration and favorable oral bioavailability, easily self-administered, have a shorter biological half-life, and less expensive than mAbs.
PD-L1 expression is regulated by several cellular signaling pathways including MAPK/ERK pathway, JAK/STAT3, PI3K/AKT pathway and by transcriptional factors like NFκB. Interestingly, these previously mentioned pathways are considered as new potential antitumor molecular targets of ATRA, major metabolite of vitamin A.
Co-targeting of more than one molecular pathway involved in carcinogenesis is evident to be superior to single pathway targeting on the level of therapeutic outcomes. In this context, the present work was undertaken to evaluate the potential effects exerted on cellular signaling pathways resulting from PD-L1 inhibition in human hepatocellular carcinoma (HCC) cell line model; HepG2. The aim was further extended to investigate the potential impact of ATRA on PD-L1 receptors expression and to demonstrate the beneficial anti-carcinogenic effect of their combination. The current study was conducted on 5 groups: Cell line I (Control group): cells received only the vehicle used for the dissolution of the drugs (0.5% DMSO) Cell line II: cells treated with 0.89205µM PD-L1 inhibitor, Cell line III: cells treated with 5.68808 µM of ATRA , Cell line IV: cells treated with 0.89205µM of PD-L1 inhibitor and 5.68808 µM of ATRA, Cell line V: cells treated with 0.03148 of PD-L1 inhibitor and 4.37256 ATRA (a reduced dose at the synergistic level obtained from DRI calculation).
The protein expression levels of PD-L, RARα, STAT3, NF-κB, pERK, pAKT, CD1, VEGF were determined by Sandwich ELISA, Caspase-3 activity was determined by colorimetric assay. Concerning molecular studies, quantitative qRT-PCR was used for the determination of the relative expression levels of the PD-L1 gene.
The results showed that three days of treatment of HepG2 cells with PD-L1 inhibitor resulted in a remarkable increase in PD-L1 gene and protein expression which were both decreased upon the addition of ATRA. Moreover, adding ATRA to the PD-L1 inhibitor resulted in a more pronounced decrease in protein expression level of STAT3, p-ERK, p-AKT, NF-κB signaling axis in parallel with a decrease in VEGF, CD1 expression and a subsequent increase in caspase-3 activity, compared to the control group. On top of that, the cells treated with ATRA showed a highly significant increase in RARα protein expression level compared to the control group.
Once again combining both drugs showed better impact highlighting the positive interaction between them. Whereas the observed combination indices CIs and dose reduction indices DRIs confirmed the synergistic effect of PD-L1 inhibitor and ATRA. Co-administration of the 2 drugs resulted in better modulatory effect on the intratumoral signaling pathways involved in PD-L1 expression, as well as more profound protection against the proliferation and angiogenic effects, in addition to the apoptotic effects. Moreover, the addition of ATRA to PD-L1 inhibitor revealed a noteworthy increase in RARα expression and abolished the elevation in PD-L1 gene and protein levels induced by PD-L1 inhibitor and could be the reason behind the limitation in its ultimate anti-tumor response. To the best of our knowledge, no previous studies investigated the influence of combined PD-L1 inhibitor and ATRA on HCC.
In summary, the combination of ATRA with PD-L1 inhibitor appeared to be efficient regarding all the studied parameters on HCC cell line. Taken together, this work provided a piece of evidence that ATRA addition to PD-L1 inhibitor therapy could add a beneficial antitumor effect.