Chemosensitization of HT29 and HT29-5FU Cell Lines by a Combination of a Multi-Tyrosine Kinase Inhibitor and 5FU Downregulates ABCC1 and Inhibits PIK3CA in Light of Their Importance in Saudi Colorectal Cancer.

Colorectal cancer (CRC) remains one of the main causes of death worldwide and in Saudi Arabia. The toxicity and the development of resistance against 5 fluorouracil 5FU pose increasing therapeutic difficulties, which necessitates the development of personalized drugs and drug combinations. OBJECTIVES: First, to determine the most important kinases and kinase pathways, and the amount of ABC transporters and KRAS in samples taken from Saudi CRC patients. Second, to investigate the chemosensitizing effect of LY294002 and HAA2020 and their combinations with 5FU on HT29, HT29-5FU, HCT116, and HCT116-5FU CRC cells, their effect on the three ABC transporters, cell cycle, and apoptosis, in light of the important kinase pathways resulting from the first part of this study. METHODS: The PamChip® peptide micro-array profiling was used to determine the level of kinase and targets in the Saudi CRC samples. Next, RT-PCR, MTT cytotoxicity, Western blotting, perturbation of cell cycle, annexin V, and immunofluorescence assays were used to investigate the effect on CRC, MRC5, and HUVEC cells. RESULTS: The kinase activity profiling highlighted the importance of the PI3K/AKT, MAPK, and the growth factors pathways in the Saudi CRC samples. PIK3CA was the most overexpressed, and it was associated with increased level of mutated KRAS and the three ABC transporters, especially ABCC1 in the Saudi samples. Next, combining HAA2020 with 5FU exhibited the best synergistic and resistance-reversal effect in the four CRC cells, and the highest selectivity indices compared to MRC5 and HUVEC normal cells. Additionally, HAA2020 with 5FU exerted significant inhibition of ABCC1 in the four CRC cells, and inhibition of PIK3CA/AKT/MAPK7/ERK in HT29 and HT29-5FU cells. The combination also inhibited EGFR, increased the preG1/S cell cycle phases, apoptosis, and caspase 8 in HT29 cells, while it increased the G1 phase, p21/p27, and apoptosis in HT29-5FU cells. CONCLUSION: We have combined the PamChip kinase profiling of Saudi CRC samples with in vitro drug combination studies in four CRC cells, highlighting the importance of targeting PIK3CA and ABCC1 for Saudi CRC patients, especially given that the overexpression of PIK3CA mutations was previously linked with the lack of activity for the anti-EGFRs as first line treatment for CRC patients. The combination of HAA2020 and 5FU has selectively sensitized the four CRC cells to 5FU and could be further studied.

Significance of Targeting VEGFR-2 and Cyclin D1 in Luminal-A Breast Cancer.

The hormonal luminal-A is the most pre-dominant sub type of breast cancer (BC), and it is associated with a high level of cyclin D1 in Saudi patients. Tamoxifen is the golden therapy for hormonal BC, but resistance of cancer cells to tamoxifen contributes to the recurrence of BC due to many reasons, including high levels of AIB1 and cyclin D1. Overcoming drug resistance could be achieved by exploring alternative targetable therapeutic pathways and new drugs or combinations. The objective of this study was to determine the differentially enriched pathways in 12 samples of Saudi women diagnosed with luminal-A using the PamChip peptide microarray-based kinase activity profiling, and to compare the activity of HAA2020 and dinaciclib with tamoxifen in singles and combinations in the MCF7 luminal-A cell line. Our results of network and pathway analysis of the 12 samples highlighted the importance of VEGFR and CDKs in promoting luminal-A breast cancer. The activation of VEGF signaling via VEGFR-2 leads to activation of PI3K/AKT kinases and an increase of cell survival, and leads to activation of Hsp90, which induces the phosphorylation of FAK1, resulting in cytoskeleton remodeling. PLC-gamma 1 is also activated, leading to FAK-2 and PKC activation. Notably, the G1/S cell cycle phases and phosphorylation processes contribute to the top seven tumorigenesis processes in the 12 samples. Further, the MTT combination of HAA2020 and dinaciclib showed the best combination index (CI), was more clonogenic against MCF7 cells compared to the other combinations, and it also showed the best selectivity index (SI) in normal MRC5 cells. Interestingly, HAA2020 and dinaciclib showed a synergistic apoptotic and G1 cell cycle effect in MCF7 cells, which was supported by their synergistic CDK2, cyclin D1, and PCNA inhibition activities. Additionally, the combination showed VEGFR-2 and Hsp90 inhibition activities in MCF7 cells. The results show the significance of targeting VEGFR-2 and cyclin D1 in Saudi luminal-A breast cancer patients, and the effect of combining HAA2020 and dinaciclib on those targets in the MCF7 model. It also warrants further preclinical and in vivo investigations for the combination of HAA2020 and dinaciclib as a possible future second-line treatment for luminal-A breast cancers.

Synergistic Anti Leukemia Effect of a Novel Hsp90 and a Pan Cyclin Dependent Kinase Inhibitors.

Acute myeloid leukemia (AML) is among the top four malignancies in Saudi nationals, and it is the top leukemia subtype worldwide. Resistance to available AML drugs requires the identification of new targets and agents. Hsp90 is one of the emerging important targets in AML, which has a central role in the regulation of apoptosis and cell proliferation through client proteins including the growth factor receptors and cyclin dependent kinases. The objective of the first part of this study is to investigate the putative Hsp90 inhibition activity of three novel previously synthesized quinazolines, which showed HL60 cytotoxicity and VEGFR2 and EGFR kinases inhibition activities. Using surface plasmon resonance, compound 1 (HAA2020) showed better Hsp90 inhibition compared to 17-AAG, and a docking study revealed that it fits nicely into the ATPase site. The objective of the second part is to maximize the anti-leukemic activity of HAA2020, which was combined with each of the eleven standard inhibitors. The best resulting synergistic effect in HL60 cells was with the pan cyclin-dependent kinases (CDK) inhibitor dinaciclib, using an MTT assay. Furthermore, the inhibiting effect of the Hsp90α gene by the combination of HAA2020 and dinaciclib was associated with increased caspase-7 and TNF-α, leading to apoptosis in HL60 cells. In addition, the combination upregulated p27 simultaneously with the inhibition of cyclinD3 and CDK2, leading to abolished HL60 proliferation and survival. The actions of HAA2020 propagated the apoptotic and cell cycle control properties of dinaciclib, showing the importance of co-targeting Hsp90 and CDK, which could lead to the better management of leukemia.

6-shogaol protects against diabetic nephropathy and cardiomyopathy via modulation of oxidative stress/NF-κB pathway.

Diabetes dramatically increases the risk of numerous heart and kidney troubles. Diabetic nephropathy (DN) and cardiomyopathy (DC) are major causes of death. The pathophysiology of DN/DC includes inflammatory and oxidative stress mechanisms. NF-κB is one of the transcription factor that mediates signal transduction processes. Nowadays, it is suggested that inhibition of NF-κB activation could delay the development of DN and DC. 6-shogaol was reported to modulate NF-κB besides its anti-oxidant and anti-inflammatory activities. Therefore, it is worth testing it against diabetic complications. Rats were divided to 4 groups: Normal control (NC), 6-shogaol (6S), diabetic control (DC), diabetic rats treated with 6-shogaol (DC+6S). BGL, BUN, serum creatinine, total urine protein, creatine kinase (CK), LDH, NO, TNF-α NF-κB were determined in serum. Heart and kidney tissues were isolated for GSH, MDA, SOD measurement and histopathology. NF-κB was estimated in kidney tissues using immunohistopathology and western blot techniques. Results showed that diabetic rats left untreated for 16 weeks showed kidney injury as evidenced from elevated BUN, serum creatinine, urine protein, TNF-α and NF-κB. Heart tissue damage was evidence from elevated CK, LDH. Diabetic rats simultaneously treated with 6-shogaol showed a protective effect on both kidney and heart as evidenced biochemically and histopathologically. Therefore, using 6-shogaol may be of value in protection against diabetic complications in kidney and heart of rats.

Structural-based design, synthesis, and antitumor activity of novel alloxazine analogues with potential selective kinase inhibition.

Protein kinases are promising therapeutic targets for cancer therapy. Here, we applied multiple approaches to optimize the potency and selectivity of our reported alloxazine scaffold. Flexible moieties at position 2 of the hetero-tricyclic system were incorporated to fit into the ATP binding site and extend to the adjacent allosteric site and selectively inhibit protein kinases. This design led to potential selective inhibition of ABL1, CDK1/Cyclin A1, FAK, and SRC kinase by 30-59%. Cytotoxicity was improved by ∼50 times for the optimized lead (10b; IC50 = 40 nM) against breast cancer (MCF-7) cells. Many compounds revealed potential cytotoxicity against ovarian (A2780) and colon carcinoma (HCT116) cells of ∼10-30 time improvement (IC50 5-17 nM). The results of the Annexin-V/PI apoptotic assay demonstrated that many compounds induced significantly early (89-146%) and a dramatically late (556-1180%) cell death in comparison to the vehicle control of MCF-7 cells. SAR indicated that 5-deazaalloxazines have a higher selectivity for Abl-1 and FAK kinases than alloxazines. The correlations between GoldScore fitness into FAK and SRC kinases and IC50 against MCF-7 and A2780 cells were considerable (R2: 0.86-0.98).