Tumor suppressor genes in the CDK-cyclin D/p16INK4a/pRb/E2F cycle have been deregulated in more than 80% of human neoplasms [15]. Thus the inhibition of CDK and induction of cell cycle arrest have been considered in cancer therapy [16]. It has been shown that in 58% of non-small cell lung carcinoma lacking INK4a expression with no change in cyclin D or CDK4 expression happened, suggesting a major role for p16INK4a in this malignancy [17]. We have found that the expression of p16 and its truncated form (i.e. p1666–156) in A549 cells induces cell death by 20–30% compared to non-transfected cells (Fig. 5 and Fig. 6). Considering the lack of endogenous expression of p16 in A549, these results indicate the role of p16 in A549 cell death. Furthermore, the cytotoxicity of irinotecan or 5FU has been increased with the expression of p16 or its truncated form in A549 cells. In cells expressing p1666–156 and treated with the IC25 of 5FU, the cytotoxicity has been comparable to the IC50 of 5FU in untransfected cells. In accordance with studies of pRb pathway in 2 types of lung cancers the following results were obtained P16 could change proportion of cells at cell cycle phases, quantity of A549 cells treating with 5FU, at sub G1 was increased when transfecting with P1666–156 and P16 full more than control cells, which shows that this constructs could arrest cell cycle [17, 18]. Cells at S phase were decreased in 5FU treated and P16 transfected cells more than control cells which were just treated with 5FU, we could see this reduction better at IC10 5FU than IC50 because higher concentration of the drug cause more cytotoxicity which could kill cells.
According to studies, the INK4a expression or function impairment plays a significant role in non-small cell lung cancer. Scientists Investigate the P16 (as the main member of INK4A family), Cyclin D and Rb expression as biomarkers for the diagnosis of non-small cell lung cancer [19, 20]. In a study performed in 2013, co-administration of an E2F inhibitor with cisplatin and gemcitabine, as well as paclitaxel on SCLC and NSCLC showed synergic effects in cell death [21].
In our study, we transfected P16 and P1666–156 to A549 cell line after transfection, P16 arrest cell cycle in G0 phase and increased cell death percentage. We demonstrated the synergic effect of P16 induction and using 5FU as chemotherapeutic agent. Another study showed an increase in the sensitivity of neck squamous cell carcinoma, which were p16-positive, to 5-FU [14].
CDK4/6 inhibitors such as palbociclib, ribociclib, and abemaciclib play an important role in inhibiting the cells' passing from the G1 phase to the S phase [22]. CDK4/6 inhibitors have shown remarkable effects on many types of solid tumors [23]. Palbociclib and abemaciclib have been approved by FDA for NSCLC [24]. Improper CDK4/6 activation is a common event in NSCLC [25]. So, the effect of CDK inhibitors on this type of cancer has been studied. The addition of a CDK inhibitor to endocrin therapy in HR + breast cancer showed an appropriate therapeutic response [26]. The combination of palbociclib and afatinib in afatinib-resistant NSCLC improved the therapeutic response and reduced afatinib resistance [27]. Cyclin D was increased in patients were treated with CDK4/6 inhibitors, to evaluate that combination of mTOR inhibitor and polbiciclib was studied. Several studies have demonstrated that combining CDK4/6 with mTOR inhibitors might reduce cancer cell proliferation in a synergistic manner [28, 29]. Single drug therapy with abemaciclib has tumor growth inhibition in many type of tumor such az NSCLC [28, 30]. Abemaciclib and gemcitabin combination have showed garater antitumor activity in lung cancer [31] as we know P16 is the main inhibitor of CDK4/6.