In vitro analysis of PI3K pathway activation genes for exploring novel biomarkers and therapeutic targets in clear cell renal carcinoma.

OBJECTIVES: The regulation of various cellular functions such as growth, proliferation, metabolism, and angiogenesis, is dependent on the PI3K pathway. Recent evidence has indicated that kidney renal clear cell carcinoma (KIRC) can be triggered by the deregulation of this pathway. The objective of this research was to investigate 25 genes associated with activation of the PI3K pathway in KIRC and control samples to identify four hub genes that might serve as novel molecular biomarkers and therapeutic targets for treating KIRC. METHODS: Multi-omics in silico and in vitro analysis was employed to find hub genes related to the PI3K pathway that may be biomarkers and therapeutic targets for KIRC. RESULTS: Using STRING software, a protein-protein interaction (PPI) network of 25 PI3K pathway-related genes was developed. Based on the degree scoring method, the top four hub genes were identified using Cytoscape's Cytohubba plug-in. TCGA datasets, KIRC (786-O and A-498), and normal (HK2) cells were used to validate the expression of hub genes. Additionally, further bioinformatic analyses were performed to investigate the mechanisms by which hub genes are involved in the development of KIRC. Out of a total of 25 PI3K pathway-related genes, we developed and validated a diagnostic and prognostic model based on the up-regulation of TP53 (tumor protein 53) and CCND1 (Cyclin D1) and the down-regulation of PTEN (Phosphatase and TENsin homolog deleted on chromosome 10), and GSK3B (Glycogen synthase kinase-3 beta) hub genes. The hub genes included in our model may be a novel therapeutic target for KIRC treatment. Additionally, associations between hub genes and infiltration of immune cells can enhance comprehension of immunotherapy for KIRC. CONCLUSION: We have created a new diagnostic and prognostic model for KIRC patients that uses PI3K pathway-related hub genes (TP53, PTEN, CCND1, and GSK3B). Nevertheless, further experimental studies are required to ascertain the efficacy of our model.

In Vivo and In Vitro Enhanced Tumoricidal Effects of Metformin, Active Vitamin D3, and 5-Fluorouracil Triple Therapy against Colon Cancer by Modulating the PI3K/Akt/PTEN/mTOR Network.

Chemoresistance to 5-fluorouracil (5-FU) is common during colorectal cancer (CRC) treatment. This study measured the chemotherapeutic effects of 5-FU, active vitamin D3 (VD3), and/or metformin single/dual/triple regimens as complementary/alternative therapies. Ninety male mice were divided into: negative and positive (PC) controls, and 5-FU, VD3, Met, 5-FU/VD3, 5-FU/Met, VD3/Met, and 5-FU/VD3/Met groups. Treatments lasted four weeks following CRC induction by azoxymethane. Similar regimens were also applied in the SW480 and SW620 CRC cell lines. The PC mice had abundant tumours, markedly elevated proliferation markers (survivin/CCND1) and PI3K/Akt/mTOR, and reduced p21/PTEN/cytochrome C/caspase-3 and apoptosis. All therapies reduced tumour numbers, with 5-FU/VD3/Met being the most efficacious regimen. All protocols decreased cell proliferation markers, inhibited PI3K/Akt/mTOR molecules, and increased proapoptotic molecules with an apoptosis index, and 5-FU/VD3/Met revealed the strongest effects. In vitro, all therapies equally induced G1 phase arrest in SW480 cells, whereas metformin-alone showed maximal SW620 cell numbers in the G0/G1 phase. 5-FU/Met co-therapy also showed the highest apoptotic SW480 cell numbers (13%), whilst 5-FU/VD3/Met disclosed the lowest viable SW620 cell percentages (81%). Moreover, 5-FU/VD3/Met revealed maximal inhibitions of cell cycle inducers (CCND1/CCND3), cell survival (BCL2), and the PI3K/Akt/mTOR molecules alongside the highest expression of cell cycle inhibitors (p21/p27), proapoptotic markers (BAX/cytochrome C/caspase-3), and PTEN in both cell lines. In conclusion, metformin monotherapy was superior to VD3, whereas the 5-FU/Met protocol showed better anticancer effects relative to the other dual therapies. However, the 5-FU/VD3/Met approach displayed the best in vivo and in vitro tumoricidal effects related to cell cycle arrest and apoptosis, justifiably by enhanced modulations of the PI3K/PTEN/Akt/mTOR pathway.

Enhanced in vitro tumoricidal effects of 5-Fluorouracil, thymoquinone, and active vitamin D3 triple therapy against colon cancer cells by attenuating the PI3K/AKT/mTOR pathway.

AIMS: This study measured the effects of 5-Fluorouracil (5-FU), calcitriol (VD3), and/or thymoquinone (TQ) single/dual/triple therapies on cell cycle progression, apoptosis, inhibition of the PI3K/AKT/mTOR pathway, and oxidative stress against colorectal cancer (CRC). MAIN METHODS: The HT29, SW480 and SW620 cell lines were treated with 5-FU (50 µM), VD3 (25 µM), and TQ (75 µM), alone or combined for 12 h, prior to cell cycle/apoptosis analyses. KEY FINDINGS: TQ monotherapy had greater anticancer effects to active VD3 or 5-FU, revealing higher expression of p21/p27/PTEN/BAX/Cyto-C/Casp-3 and increased levels of total glutathione, with inhibitions in CCND1/CCND3/BCL-2 and PI3K/AKT/mTOR molecules, alongside higher rates of apoptosis in HT29, SW480 and SW620 cells (P < 0.005 for all markers). Additionally, all combination protocols revealed enhanced modulations of the PI3K/PTEN/Akt/mTOR pathway, higher expression of p21/p27/PTEN/BAX/Cyto-C/Casp-3, and better anti-oxidant effects, than the monotherapies. Although TQ/5-FU and TQ/VD3 co-therapies were better relative to the VD3/5-FU regimen, the best tumoricidal effects were observed with triple therapy in the HT29 and SW480 cell lines, possibly by boosted attenuations of the PI3K/AKT/mTOR oncogenic pathway. In contrast, TQ single treatment was more effective than the triple therapy regimen in metastatic SW620 cells, suggesting that this protocol would be more useful therapeutically in late-stage CRC. SIGNIFICANCE: In conclusion, this study is the first to demonstrated enhanced anti-tumorigenic effects for VD3, TQ, and 5-FU triple therapy against CRC cells and could represent the best strategy for treating early stages of malignancy, whereas TQ monotherapy could be a better approach for treating metastatic forms of the disease.

Profiling Activins and Follistatin in Colorectal Cancer According to Clinical Stage, Tumour Sidedness and Smad4 Status.

This study explored the roles of activins and follistatin in colorectal cancers. Paired malignant and normal colonic tissues were collected from archived paraffin-embedded (n = 90 patients) alongside fresh (n = 40 patients) specimen cohorts. Activin ß-subunits, follistatin and Smad4 mRNAs and proteins were measured by real-time PCR and immunohistochemistry (IHC). Mature activin-A, -B, -AB and follistatin proteins were measured by ELISA. Cancer tissues having ≤ the 20th percentile of the Smad4 IHC score were considered as low (L-S4) group. The Smad4-intact SW480 and Smad4-null HT29 colon cancer cell lines were treated with activins and follistatin, and cell cycle was analysed by flow cytometry. The cell cycle inducing (CCND1/CCND3) and inhibitory (p21/p27) proteins alongside the survival (survivin/BCL2) and pro-apoptosis (Casp-8/Casp-3) markers were measured by immunofluorescence. Thirty-nine patients had right-sided cancers (30%) and showed higher rates of L-S4 tumours (n = 17; 13.1%) alongside worse clinicopathological characteristics relative to left-sided cancers. The ßA-subunit and activin-A increased, whilst ßB-subunit and activin-AB decreased, in malignant sites and the late-stage cancers revealed the greatest abnormalities. Interestingly, follistatin declined markedly in early-stage malignant tissues, whilst increased significantly in the advanced stages. All activin molecules were comparable between the early stage right- and left-sided tumours, whereas the late-stage right-sided cancers and L-S4 tumours showed more profound deregulations. In vitro, activin-A increased the numbers of the SW480 cells in sub-G1 and G0/G1-phases, whereas reduced the HT29 cell numbers in the sub-G1 phase with simultaneous increases in the G0/G1 and S phases. The p21/p27/Casp-8/Casp-3 proteins escalated, whilst CCND1/CCND3/BCL2/survivin declined in the SW480 cells following activin-A, whereas activin-A only promoted p21 and p27 alongside reduced CCND3 in the HT29 cells. By contrast, activin-AB increased the numbers of SW480 and HT29 cells in Sub-G1 and G0/G1-phases and promoted the anti-cancer and reduced the oncogenic proteins in both cell lines. In conclusion, activins and follistatin displayed stage-dependent dysregulations and were markedly altered during the advanced stages of right-sided and L-S4 cancers. Moreover, the activin-A actions in CRC could be Smad4-dependent, whereas activin-AB may act as a Smad4-independent tumour suppressor protein.

Chemopreventive effects of vitamin D3 and its analogue, paricalcitol, in combination with 5-fluorouracil against colorectal cancer: The role of calcium signalling molecules.

BACKGROUND: Although vitamin D (VD) is chemoprotective and enhances 5-fluorouracil (5-FU) cytotoxicity against colorectal cancer (CRC), little is known about its potential calcium (Ca2+)-mediated anti-tumorigenic actions. Therefore, this study compared between VD and its non-calcaemic analogue, Paricalcitol (Pcal), ± 5-FU in relation to chemoprevention and Ca2+-mediated apoptosis in vivo and in vitro. METHODS: Seventy male mice were distributed to: negative controls, positive controls (PC), VD, Pcal, 5-FU, VD + 5-FU and Pcal+5-FU groups. All groups, except negative, received two consecutive azoxymethane (AOM)-injections (10 mg/Kg/week) for CRC induction. VD3 (1000 IU/kg; three times/week) and Pcal (1.25 µg/kg; three times/week) injections started week-16 post-AOM and for 10 weeks. Three successive 5-FU cycles began at week-21 (50 mg/Kg/week). Similar protocols with VD3, Pcal and/or 5-FU were applied in the HT29 colon cancer cells. RESULTS: The PC group had abundant malignant tumours, markedly elevated proliferation markers (survivin/CCND1) and declines in cyclin-dependent kinase-inhibitor-1A, pro-apoptotic molecules (p53/BAX/cytochrome_C/caspase-3), tissue Ca2+ concentrations and Ca2+-dependent proteins (CaSR/CAM/CAMKIIA). All monotherapies equally reduced tumour numbers and proliferation markers whilst promoting the anti-tumorigenic molecules. VD and/or 5-FU, but not Pcal monotherapy, enhanced Ca2+ levels and Ca2+-related molecules (CaSR/CAM/CAMKIIA/BAX/cytochrome_C) in vivo and in vitro. However, VD + 5-FU co-therapy showed the lowest tumour numbers, the highest cell numbers in sub-G1 phase of cell cycle, alongside the most effective modulations of oncogenes, tumour suppressors and Ca2+-related molecules at the gene and protein levels in vivo and in vitro. CONCLUSIONS: VD3 was superior than Paricalcitol in potentiating 5-FU cytotoxicity, possibly by upregulating several Ca2+-related molecules involved in tumour suppression.