CC is one of the most common types of cancers in women, and for advanced CC, the median survival is a mere 16.8 months [6]. Immunotherapy has been a successful treatment method in several types of malignancies including melanoma, renal, and lung cancer, yet there is little research on investigating the use of immunotherapy for the treatment of CC. Our research examines the treatment of CC cells with IL-29, and it was found that IL-29 resulted in decreased proliferation and increased apoptosis of CC cells. Further expanding into the possible mechanisms, RT-PCR and IHC were used to investigate the levels of anti-proliferative and pro-apoptotic molecules. Our research uncovered that IL-29 treated CC cells expressed upregulated levels of anti-proliferative molecules p18 and p27, as well as upregulated levels of pro-apoptotic molecule TRAILR1.

Chemoradiotherapy became the standard treatment regimen for locally advanced CC in the 1990s which provided large improvements in survival. Unfortunately, recent attempts to improve chemoradiotherapy have had many setbacks [4, 29]. Recent investigation of immunotherapy based treatment of many cancers holds promise that this treatment option could be used to fight CC. CC is highly associated with high-risk HPV subtypes, and the integration of HPV E6 and E7 into the genome result in tumor cell survival and immune escape [30, 31]. The cell cycle and apoptotic pathways are key hallmarks of cancer cell evasion of death and unrelenting survival, thus investigation of the molecules involved in the pathways are crucial to developing immunotherapy for CC. Our unique research examines the use of IL-29 as a treatment option for CC, and we further examine the influence IL-29 has on anti-proliferative and pro-apoptotic molecules. Given the properties of the immune system involvement in the progression of CC through HPV infection, targeted immunotherapy of CC may provide improved survival.

One of the six major hallmarks of neoplasms are unregulated cell growth, and this relates to uncontrolled cellular replication in the cell cycle. The cell cycle involves many positive and negative regulators to keep replication at a delicate balance, however, cancer infamously is characterized by a loss of this balance in favor of an excess of cell growth. Two major suppressors of the cell cycle are p18 and p27 [32]. P18 has been shown to have anti-tumor behaviors; for example a deficiency of p18 predisposes humans to hematopoietic lymphoid malignancies, including T-cell leukemia and multiple myeloma [32]. In similar research, inactivating mutations of p18 occurred with activating RET mutations in human medullary thyroid carcinoma and pheochromocytoma, indicating that p18 functions as a tumor suppressor [33].

P27 is another tumor suppressor that acts to inhibit cyclin-CDK complexes resulting in arrest of the cell cycle. In prostate cancer, increased levels of E6-associated protein (E6AP) are seen, and one study determined that E6AP is responsible for silencing p27 [34]. In evaluation of p27 and colon adenocarcinoma, p27 was found to be inversely correlated with metastasis to lymph nodes [35]. Thus not only is p27 important in the initial formation of cancer, it is also highly related to the metastatic potential of malignancies. Our study demonstrates that treatment of CC with IL-29 resulted in decreased growth and proliferation of cancer cells. We subsequently discovered that the levels of both anti-proliferative molecules p18 and p27 were elevated in CC cells. Based on our findings, we propose the increased levels of p18 and p27 post-treatment with IL-29 in CC cells is part of the mechanism behind decreased cervical neoplasm growth and proliferation. From this, one can further develop targeted immunotherapies that manipulates this pathway for treatment of CC.

Interestingly, our results demonstrated an increase in mRNA expression of pro-proliferative molecules cyclin B, cyclin D, and CDK4 in SiHa CC cells treated with IL-29. We propose that this increase in pro-proliferative molecules is a compensatory effect, and it is a direct response to the growth inhibition of IL-29 on CC cells. Thus, with the increase in p18 and p27 promoting an anti-growth cancer cell environment, the CC cells attempt to restore balance by increasing pro-proliferative molecules. However as shown, the overall effect is inhibited growth of CC cells that were treated with IL-29, and the increase in pro-proliferative molecules is simply a response to decreased cellular growth.

An additional cellular process that is out of balance in tumorous growths is the apoptotic pathway. Apoptosis is defined as programmed cell death, and cancer cells have many ways to manipulate the avoidance of apoptosis to increase their survival [36]. Ligands of the TNF family are involved in apoptosis in a p53 independent mechanism, and thus are important in that p53 mutations are often present in cancer. These ligands bind to the TRAIL receptors, resulting in receptor aggregation and formation of the death inducing signaling complex which ultimately leads to apoptosis. In recent reports, the TRAILR1 receptor has the most prominent role, especially in lymphoid malignancies and leukemic cells [37,38,39]. Additionally, TRAILR1 is able to trigger apoptosis in the deadly pancreatic cell line [40]. Kretz et al., conducted a review covering the topic of manipulating the TRAIL pathway as an anticancer approach for pancreatic cancer. Unfortunately, clinical trials involving use of agonistic antibodies to TRAILR1 in pancreatic cancer have been disappointing [17]. It is proposed that there exists a type of TRAIL resistance in tumor cells, and this may be contributing to the lack of clinical evidence of the effectiveness of TRAILR1 agonistic antibodies. The mechanism of the TRAIL/TRAIL receptor induced apoptosis is an area that should be continually research because there exists such a potential in using this pathway to increase cancer cell apoptosis. In our study, IL-29 treatment was associated with increased apoptosis of CC cells, and alongside this finding, TRAILR1 was found to be upregulated. We propose that the possible mechanism behind IL-29 induced CC cell apoptosis is related to the increased expression of the TRAILR1. Our hope is that in future studies, this relationship could be further evaluated to assist in developing a molecular target for immunotherapy that mimics these results.

Interestingly, our results demonstrate an increase in mRNA expression of anti-apoptotic molecule cellular FLICE inhibitory protein (c-FLIP) in SiHa CC cells treated with IL-29. We propose that this increase in anti-apoptotic molecule is again a compensatory effect, similar to that seen with the proliferative molecules. After treatment with IL-29, we believe the increase in TRAILR1 promotes increased apoptosis in the cancer cell microenvironment. Then, to offset this increased apoptotic effect, the pro-apoptotic molecule c-FLIP increases. The increase in c-FLIP is simply a response to increased apoptosis in the microenvironment however, no significant difference was found in the expression of P21, P53, cyclin D, and CDK 2 by IL-29, suggesting that these molecules were not significant contributors to the mechanism by which IL-29 inhibits growth of SiHa cervical cancer cells.

It is necessary to point out that there are some limitations in this study. We only presented the results using SiHa CC cell line. Interestingly, the growth inhibitory effect of IL-29 on CC was also found when using a different CC cell line HeLa, suggesting that our findings are true of CC in general and are not specific only to SiHa CC cells. We plan to present our study in HeLa cells in a separate manuscript. In this study, due to limited available slides, only upregulation of p27 and TRAILR1 mRNA, but not p21 mRNA, were further confirmed by IHC. In fact, this study was not conducted on animals, so these effects have not yet been examined in vivo. As discussed, CC is a deadly gynecological cancer, however, there are many other types of female cancer that needs better treatment options explored. Western blot was not used in this study as a method to measure proteins, in our past study, we found Western blot to be consistent with mRNA and IHC data; therefore, we did not believe it to be necessary to use Western blot in this study. Our study focused on the interleukin IL-29. There are numerous interleukins that influence the pathogenesis of cervical cancer, so we believe future experiments could examine other interleukins. Our study concluded that IL-29 upregulated p18, p27, and TRAILR1, and it would be of interest to investigate if silencing p18, p27, and TRAILR1 by SiRNA technique could reverse the inhibitory effect of IL-29 on the growth and apoptosis of CC cells.

In summary, CC treated with IL-29 were found to have decreased growth and proliferation and increased apoptosis. Our study examined the possible molecular mechanisms behind this relationship by conducting RT-PCR and IHC. With these methods, it was found that SiHa CC cells treated with IL-29 had upregulated expression of key anti-proliferative molecules p18 and p27 and pro-apoptotic molecule TRAILR1. Our work is the first to discover the powerful anticancer action of IL-29 in CC. CC is a devastating diagnosis for women, and current treatment methods need improved to increase survival rates. Interleukin based immunotherapy for the treatment of CC holds promise for the gynecologic-oncology field, and our study is the first to contribute this key information.