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Abstract Background The incidence rate of non-small cell lung cancer (NSCLC) has been increasing worldwide, and the correlation of circadian rhythm disruption with a raised risk of cancer and worse prognosis has been shown by accumulating evidences recently. On the other hand, drug resistance and the impact of tumor heterogeneity have been inevitable in NSCLC therapy. These both lead to an urgent need to identify more useful prognostic and predictive markers for NSCLC diagnosis and treatment, especially on the aspect of circadian clock genes. Methods The expression of the main clock genes in cancer was probed with TIMER and Oncomine databases. The prognostic value of key clock genes was probed systematically with the Kaplan–Meier estimate and Cox regression on samples from TCGA database. RT-qPCR was performed on patient tissue samples to further validate the results from databases. The functional enrichment analysis was performed using the “ClusterProfiler” R package, and the correlation of key clock genes with tumor mutation burden, immune checkpoint, and immune infiltration levels were also assessed using multiple algorithms including TIDE, TIMER2.0, and XCELL. Results TIMELESS was significantly upregulated in lung tissue of clinical lung cancer patients as well as TCGA and Oncomine databases, while RORA was downregulated. Multivariate Cox regression analysis indicated that TIMELESS (P = 0.004, HR = 1.21 [1.06, 1.38]) and RORA (P = 0.047, HR = 0.868 [0.755, 0.998]) has a significant correlation with overall survival in NSCLC. Genes related to TIMELESS were enriched in the cell cycle and immune system, and the function of RORA was mainly focused on oncogenic signaling pathways or glycosylation and protein activation. Also, TIMELESS was positively correlated with tumor mutation burden while RORA was negatively correlated with it. TIMELESS and RORA were also significantly correlated with immune checkpoint and immune infiltration levels in NSCLC. Additionally, TIMELESS showed a significant positive relationship with lipid metabolism. Conclusions TIMELESS and RORA were identified as key clock genes in NSCLC, and were independent prognostic factors for overall survival in NSCLC. The function of them were assessed in many aspects, indicating the strong potential of the two genes to serve as biomarkers for NSCLC progression and prognosis.

Background Pancreatic cancer is one of the most aggressive human malignancies with a extremely low 5-year survival rate. Hence, the search for more effective anti-pancreatic cancer agents is urgent. Methods PaTu8988 pancreatic cancer cells were treated with different concentrations of suberoylanilide hydroxamic acid (SAHA), cell survival, proliferation, migration and vasculogenic mimicry (VM) were analyzed. Associated signaling changes were also analyzed by RT-PCR and Western blots. Results Here, we reported that SAHA, a histone deacetylase inhibitor (HDACi), exerted significant inhibitory efficiency against pancreatic cancer cell survival, proliferation, migration and VM. SAHA dose-dependently inhibited PaTu8988 pancreatic cancer cell growth with the IC-50 of 3.4 ± 0. 7 µM. Meanwhile, SAHA suppressed PaTu8988 cell cycle progression through inducing G2/M arrest, which was associated with cyclin-dependent kinase 1 (CDK-1)/cyclin-B1 degradation and p21/p27 upregulation. Further, SAHA induced both apoptotic and non-apoptotic death of PaTu8988 cells. Significantly, SAHA suppressed PaTu8988 cell in vitro migration and cell-dominant tube formation or VM, which was accompanied by semaphorin- 4D (Sema-4D) and integrin-β5 down-regulation. Our evidences showed that Akt activation might be important for Sema-4D expression in PaTu8988 cells, and SAHA-induced Sema-4D down-regulation might be associated with Akt inhibition. Conclusions This study is among the first to report the VM formation in cultured human pancreatic cancer cells. And we provided strong evidence to suggest that SAHA executes significant anti-VM efficiency in the progressive pancreatic cancer cells. Thus, SAHA could be further investigated as a promising anti-pancreatic cancer agent. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via the original article.

Abstract Background We prepared an anti-p21Ras scFv which could specifically bind with mutant and wild-type p21Ras. However, it cannot penetrate the cell membrane, which prevents it from binding to p21Ras in the cytoplasm. Here, the RGD4C peptide was used to mediate the scFv penetration into tumor cells and produce antitumor effects. Methods RGD4C-EGFP and RGD4C-p21Ras-scFv recombinant expression plasmids were constructed to express fusion proteins in E. coli, then the fusion proteins were purified with HisPur Ni-NTA. RGD4C-EGFP was used as reporter to test the factors affecting RGD4C penetration into tumor cell. The immunoreactivity of RGD4C-p21Ras-scFv toward p21Ras was identified by ELISA and western blotting. The ability of RGD4C-p21Ras-scFv to penetrate SW480 cells and colocalization with Ras protein was detected by immunocytochemistry and immunofluorescence. The antitumor activity of the RGD4C-p21Ras-scFv was assessed with the MTT, TUNEL, colony formation and cell migration assays. Chloroquine (CQ) was used an endosomal escape enhancing agent to enhance endosomal escape of RGD4C-scFv. Results RGD4C-p21Ras-scFv fusion protein were successfully expressed and purified. We found that the RGD4C fusion protein could penetrate into tumor cells, but the tumor cell entry of was time and concentration dependent. Endocytosis inhibitors and a low temperature inhibited RGD4C fusion protein endocytosis into cells. The change of the cell membrane potential did not affect penetrability. RGD4C-p21Ras-scFv could penetrate SW480 cells, effectively inhibit the growth, proliferation and migration of SW480 cells and promote this cells apoptosis. In addition, chloroquine (CQ) could increase endosomal escape and improve antitumor activity of RGD4C-scFv in SW480 cells. Conclusion The RGD4C peptide can mediate anti-p21Ras scFv entry into SW480 cells and produce an inhibitory effect, which indicates that RGD4C-p21Ras-scFv may be a potential therapeutic antibody for the treatment of ras-driven cancers.

Abstract Background Colorectal cancer (CRC) is the most common type of gastrointestinal cancer. CRC gene therapy mediated by adenovirus holds great promise for the treatment of malignancies. However, intravenous delivery of adenovirus exhibits limited anti-tumor activity in vivo when used alone. Methods In this study, the antitumor activity of the recombinant adenovirus KGHV500 was assessed with the MTT, TUNEL, Matrigel invasion and cell migration assays. To enhance the intravenous delivery of KGHV500 in vivo, cytokine-induced killer (CIK) cells were used as a second vector to carry KGHV500. We explored whether CIK cells could carry the recombinant adenovirus KGHV500 containing the anti-p21Ras single chain fragment variable antibody (scFv) gene into tumors and enhance antitumor potency. Results Our results showed that KGHV500 exhibited significant antitumor activity in vitro. In the nude mouse SW480 tumor xenograft model, the combination of CIK cells with KGHV500 could induce higher antitumor activity against colorectal cancer in vivo than that induced by either CIK or KGHV500 alone. After seven days of treatment, adenovirus and scFv were detected in tumor tissue but were not detected in normal tissues by immunohistochemistry. Therefore, KGHV500 replicates in tumors and successfully expresses anti-p21Ras scFv in a colorectal cancer xenograft model. Conclusions Our study provides a novel strategy for the treatment of colorectal cancer by combining CIK cells with the recombinant adenovirus KGHV500 which carried anti-p21 Ras scFv.