Discovery of Selective and Potent Macrocyclic CDK9 Inhibitors for the Treatment of Osimertinib-Resistant Non-Small-Cell Lung Cancer.

Effectiveness of epidermal growth factor receptor (EGFR) inhibitors, including Osimertinib, for treating non-small-cell lung cancer (NSCLC) is limited due to the continuous emergence of drug resistance. Hence, it is urgent to develop new therapeutic approaches. CDK9, a key regulator of RNA transcription, has emerged as a promising target for the development of antitumor drugs due to its crucial role in modulating the levels of antiapoptotic protein Mcl-1. Herein, we present the synthesis, optimization, and evaluation of selective CDK9 inhibitors with a macrocyclic scaffold that effectively suppresses the growth of NSCLC cells. Notably, compound Z11, a potent CDK9 inhibitor (IC50 = 3.20 nM) with good kinase selectivity, significantly inhibits cell proliferation and colony formation and induces apoptosis in Osimertinib-resistant H1975 cells. Furthermore, Z11 demonstrates a significant suppression of tumor growth in six patient-derived organoids, including three organoids resistant to Osimertinib. Overall, Z11 served as a promising macrocycle-based CDK9 inhibitor for treating Osimertinib-resistant NSCLC.

Design and optimization of selective and potent CDK9 inhibitors with flavonoid scaffold for the treatment of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a prevalent hematological tumor associated with a high morbidity and mortality rate. CDK9, functioning as a pivotal transcriptional regulator, facilitates transcriptional elongation through phosphorylation of RNA polymerase II, which further governs the protein levels of Mcl-1 and c-Myc. Therefore, CDK9 has been considered as a promising therapeutic target for AML treatment. Here, we present the design, synthesis, and evaluation of CDK9 inhibitors bearing a flavonoid scaffold. Among them, compound 21a emerged as a highly selective CDK9 inhibitor (IC50 = 6.7 nM), exhibiting over 80-fold selectivity towards most other CDK family members and high kinase selectivity. In Mv4-11 cells, 21a effectively hindered cell proliferation (IC50 = 60 nM) and induced apoptosis by down-regulating Mcl-1 and c-Myc. Notably, 21a demonstrated significant inhibition of tumor growth in the Mv4-11 xenograft tumor model. These findings indicate that compound 21a holds promise as a potential candidate for treating AML.

Prognostic Value of the Platelet-to-Lymphocyte Ratio in Patients With Melanoma: A Meta-Analysis.

Background: The prognostic role of the platelet-to-lymphocyte ratio (PLR) is controversial in patients with melanoma. Therefore, we performed a meta-analysis to assess the prognostic value of the PLR in patients with melanoma. Methods: PubMed, Web of Science, Embase, Cochrane library, WanFang, and China National Knowledge Infrastructure were searched for eligible studies. Pooled hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated to evaluate the association between the PLR and overall survival (OS) and progression-free survival (PFS). Results: Nine studies with 2,396 patients were included in this meta-analysis. A high PLR was a predictor of shorter OS (HR = 1.67, 95% CI = 1.18-2.38, p = 0.004), but not PFS (HR = 1.53, 95% CI = 0.96-2.44, p = 0.075) in patients with melanoma. Subgroup analysis revealed that the PLR remained a significant prognostic indicator of both OS and PFS in patients with non-metastatic disease; the PLR cutoff value of <120 had a consistent prognostic value. Conclusions: An increased PLR was associated with poor OS of patients with melanoma. Hence, we suggest that the preoperative PLR could be used to identify high-risk patients and provide information regarding the prognosis of patients with melanoma.

Prognostic and clinicopathological utility of programmed death-ligand 1 in malignant pleural mesothelioma: A meta-analysis.

OBJECTIVE: Programmed death ligand 1 (PD-L1) has been reported to be connected to prognosis in individuals with malignant pleural mesothelioma (MPM), although there is no consensus based on data from previous studies. Accordingly, this quantitative meta-analysis investigated prognostic and clinicopathological utility of PD-L1 in patients with MPM. METHODS: A comprehensive search of the PubMed, Web of Science, Embase, and Cochrane Library databases for articles published up to October 4, 2019 was performed. Studies using immunohistochemical techniques to detect/quantify the expression of PD-L1 in MPM tissue were enrolled in the analysis. The combined hazard ratio (HR) and corresponding 95% confidence interval (CI) was applied to assess the association between PD-L1 expression and overall survival (OS). RESULTS: A total of 11 studies comprising 1606 patients was included in the present meta-analysis. For OS, pooled data revealed an HR of 1.50 (95% CI 1.32-1.70; p < 0.001), suggesting that patients with PD-L1 overexpression experience inferior OS. Subgroup analysis revealed that elevated PD-L1 remained a significant prognostic indicator for worse OS, irrespective of sample size, cut-off value, ethnicity, and Newcastle-Ottawa Scale score. Moreover, PD-L1 overexpression was associated with non-epithelioid histology (odds ratio 4.30 [95% CI 1.89-9.74]; p < 0.001). CONCLUSIONS: Results of this meta-analysis show that elevated expression of PD-L1 could be a factor predicting poorer survival in patients with MPM.

Excision repair cross-complementing group 1 (ERCC1) overexpression inhibits cell apoptosis and is associated with unfavorable prognosis of esophageal squamous cell carcinoma.

Excision repair cross-complementing group 1 (ERCC1) functions as a nucleotide excision repair (NER) enzyme. Altered ERCC1 expression or function is closely associated with cancer development and progression. This study determined the association of ERCC1 expression with survivin expression, clinicopathological characteristics, and survival of esophageal squamous cell carcinoma (ESCC) patients after postoperative concurrent chemoradiotherapy.Tissue specimens from 102 resected ESCC patients were acquired for immunohistochemical analysis of ERCC1 and survivin protein expression.ERCC1 expression was detected in 62.7% of ESCC tissues and in 9.8% of normal squamous epithelium tissues (P < .01), while survivin expression was detected in 60.8% of ESCC tissues and in 19.6% of normal squamous epithelia (P < .01). ERCC1 overexpression associated with advanced tumor clinical stage and lymph node metastasis (P < .05), but not with tumor size, depth of invasion, or differentiation (P > .05). ERCC1 overexpression was also associated with survivin levels (r = 0.42, P < .01) and worse progression-free survival of ESCC patients after concurrent chemoradiotherapy. Multivariate analysis data revealed that ERCC1 and survivin protein expression were independent predictors of overall survival of ESCC patients after chemotherapy and/or radiotherapy (P < .05).ERCC1 overexpression is an important phenotype that is associated with ESCC lymph node metastasis and advanced tumor clinical stages. ERCC1 expression may also inhibit ESCC cell apoptosis via regulating survivin expression, and ERCC1 and survivin overexpression are independent predictors of prognosis for ESCC patients who receive chemotherapy and/or radiotherapy.

MicroRNA-92a Promotes Colorectal Cancer Cell Growth and Migration by Inhibiting KLF4.

Colorectal cancer (CRC) is the third most common malignancy with high mortality around the world. However, the biological mechanism of CRC carcinogenesis is not completely known. In the present study, we determined the role of miR-92a in the regulation of CRC cell motility. Expression of miR-92a is aberrantly upregulated in human CRC tissues and cultured cells, as shown by RT-PCR analysis. The effects of miR-92a on the proliferation and migration of human CRC SW620 and LoVo cells were measured by CCK-8 and Transwell assay, respectively. Results showed that the proliferation and migration capacity of both SW620 and LoVo cells were significantly increased by miR-92a mimic transfection but reduced by miR-92a inhibition. Additionally, KLF4 was identified as a direct target of miR-92a in CRC cells through bioinformatics and luciferase reporter analysis. KLF4 overexpression attenuated the effects of miR-92a on the regulation of CRC cell motility. Further studies suggested that the cell cycle inhibitor p21 was aberrantly downregulated in CRC cells, whereas this inhibition was reversed by miR-92a inhibitor. In conclusion, our data demonstrated that miR-92a may play a positive role in the colorectal carcinogenesis by promoting the proliferation and migration of CRC cells through targeting KLF4 as well as downstream p21. This could be an alternative therapeutic target for CRC.

Preclinical Evaluation of Liposomal C8 Ceramide as a Potent anti-Hepatocellular Carcinoma Agent.

Hepatocellular carcinoma (HCC) remains a global health threat. The search for novel anti-HCC agents is urgent. In the current study, we synthesized a liposomal C8 ceramide, and analyzed its anti-tumor activity in pre-clinical HCC models. The liposomal C8 (ceramide) potently inhibited HCC cell (HepG2, SMMC-7721 and Huh-7 lines) survival and proliferation, more efficiently than free C8 ceramide. Yet, non-cancerous HL7702 human hepatocytes were resistant to the liposomal C8 treatment. Liposomal C8 activated caspase-dependent apoptosis in HCC cells, and HCC cytotoxicity by liposomal C8 was significantly attenuated with co-treatment of caspase inhibitors. At the molecular level, we showed that liposomal C8 activated ASK1 (apoptosis signal-regulating kinase 1)-JNK (Jun N-terminal protein kinase) signaling in HCC cells. On the other hand, JNK pharmacological inhibition or dominant negative mutation, as well as ASK1 shRNA-knockdown remarkably inhibited liposomal C8-induced apoptosis in HCC cells. Further studies showed that liposomal C8 inhibited AKT-mTOR (mammalian target of rapamycin) activation in HCC cells. Restoring AKT-mTOR activation by introducing a constitutively-active AKT alleviated HepG2 cytotoxicity by liposomal C8. In vivo, intravenous (i.v.) injection of liposomal C8 significantly inhibited HepG2 xenograft growth in severe combined immuno-deficient (SCID) mice, and mice survival was significantly improved. These preclinical results suggest that liposomal C8 could be further studied as a valuable anti-HCC agent.

Inhibition of janus kinase 2 by compound AG490 suppresses the proliferation of MDA-MB-231 cells via up-regulating SARI (suppressor of AP-1, regulated by IFN).

OBJECTIVES: The Janus kinase-signal transducers and activators of transcription signaling pathway (JAK/STAT pathway) play an important role in proliferation of breast cancer cells. Previous data showed that inhibition of STAT3 suppresses the growth of breast cancer cells, but the associated mechanisms are not well understood. This study aims to investigate the effect and associated mechanisms of JAK/STAT pathway inhibitor AG490 on proliferation and suppression of breast cancer cells. MATERIALS AND METHODS: CCK-8 assay and trypan blue exclusion assay were used to investigate the cytotoxicity of AG490 to MDA-MB-231 cells. Real-time PCR was used to detect the mRNA level of SARI (suppressor of AP-1, regulated by IFN). Western blot was used to analyze the protein levels of SARI, phospho-STAT3 and total STAT3. Luciferase reporter assay was adopted to explore the mechanism of SARI mRNA upregulation. RESULTS: AG490 suppressed the proliferation of MDA-MB-231 cells in a dose-dependent manner. AG490 significantly up-regulated the mRNA and protein levels of SARI in MDA-MB-231 cells. Knockdown of SARI obviously attenuated AG490-induced growth suppression effect in MDA-MB-231 cells. Furthermore, AG490 dramatically enhanced the transcription activity of SARI promoter. But the transcription activity of truncated SARI promoter, which does not contain STAT3 binding site, cannot be activated by AG490 treatment. CONCLUSION: We demonstrate in this study that AG490 suppresses the proliferation of MDA-MB-231 cells through transcriptional activation of SARI.

Curcumin-piperine mixtures in self-microemulsifying drug delivery system for ulcerative colitis therapy.

Curcumin (CUR) is a poorly water-soluble drug and its absorption is very low. In this study, CUR and piperine (PIP) were co-encapsulated into the nanoformulation called self-microemulsifying drug delivery system (SMEDDS) to improve the stability and water-solubility of CUR and enhance its anti-colitis activity. The formulation of CUR-PIP-SMEDDS was prepared to encapsulate two hydrophobic components CUR and PIP, and then was characterized by assessing appearance, morphology, particle size, zeta potential and drug encapsulation efficiency. The appearance of CUR-PIP-SMEDDS remained clarified and transparent, and the microemulsion droplets appeared spherical without aggregation. The mean size of microemulsion droplet formed from CUR-PIP-SMEDDS was 15.87 ± 0.76 nm, and the drug encapsulation efficiency of SMEDDS for CUR and PIP were (94.34 ± 2.18)% and (90.78 ± 2.56)%, respectively. The vitro stability investigation of CUR-PIP-SMEDDS in colon tissue suggested that using SMEDDS as a delivery vehicle and co-encapsulated with PIP, CUR was more stable than drug solution in colons site. Meanwhile, the anti-inflammatory activity of CUR-PIP-SMEDDS was evaluated on DSS-induced colitis model. The results showed that CUR-PIP-SMEDDS exhibited definite anti-colitis activity by directing CUR-PIP-SMEDDS to inflammatory colon tissue through retention enema administration. Our study illustrated that the developed CUR-PIP-SMEDDS formulation was a potential carrier for developing colon-specific drug delivery system of CUR for ulcerative colitis treatment.

Robinetinidol-flavone attenuates cholesterol synthesis in hepatoma cells via inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

Robinetinidol-(4ß,2')-tetrahydroxy-flavone (RBF) is an oligomeric condensed polyphenol that has been shown to exhibit anti-obesity effects in mice. However, little is know regarding its effect on cholesterol synthesis. The present study therefore aimed to investigate the effect of RBF on cholesterol synthesis. It was determined that RBF decreased serum total cholesterol and low density lipoprotein cholesterol in rats by 25.9 and 50.8%, respectively (P<0.001). These results strengthen evidence for the hypothesis that RBF exerts anti-atherogenic effects in vivo. Furthermore, RBF decreased cholesterol synthesis by 72%, when measured using a 3 h period of radiolabeled acetate incorporation into cholesterol, but not when using radiolabelled mevalonate, suggesting that RBF-mediated inhibition occurred largely at or above the level of 3-hydroxy-3-methylglutaryl-coenzymeA (HMG-CoA) reductase. The mechanism by which RBF inactivates HMG-CoA reductase may be attributed to the induction of phosphorylation of adenosine monophosphate (AMP)-kinase, since these results showed that RBF increased phosphorylation of AMP-kinase and HMG-CoA reductase by 2.1- and 3.2-fold, respectively, within 30 min of addition. These results suggest that RBF may be a potential therapeutic agent for hypercholesteremia.

Coexistence of hyperlipidemia and acute cerebral ischemia/reperfusion induces severe liver damage in a rat model.

AIM: To investigate the correlation of hyperlipemia (HL) and acute cerebral ischemia/reperfusion (I/R) injury on liver damage and its mechanism. METHODS: Rats were divided into 4 groups: control, HL, I/R and HL+I/R. After the induction of HL via a high-fat diet for 18 wk, middle cerebral artery occlusion was followed by 24 h of reperfusion to capture I/R. Serum alanine transaminase (ALT) and aspartate aminotransferase (AST) were analyzed as part of liver function tests and liver damage was further assessed by histological examination. Hepatocyte apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. The expression of genes related to apoptosis (caspase-3, bcl-2) was assayed by immunohistochemistry and Western blotting. Serum tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1) and liver mitochondrial superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA) and Ca(2+) levels were measured to determine inflammatory and oxidative/antioxidative status respectively. Microsomal hydroxylase activity of the cytochrome P450 2E1 (CYP2E1)-containing enzyme was measured with aniline as the substrate, and CYP2E1 expression in the liver tissue and microsome was determined by immunohistochemistry and Western blotting respectively. RESULTS: HL alone induced by high-fat diet for 18 wk resulted in liver damage, indicated by histopathological analysis, and a considerable increase in serum ALT (25.13 ± 16.90 vs 9.56 ± 1.99, P < 0.01) and AST levels (18.01 ± 10.00 vs 11.33 ± 4.17, P < 0.05) compared with control. Moreover, HL alone induced hepatocyte apoptosis, which was determined by increased TUNEL-positive cells (4.47 ± 0.45 vs 1.5 ± 0.22, P < 0.01), higher caspase-3 and lower bcl-2 expression. Interestingly, compared with those in control, HL or I/R groups, massive increases of serum ALT (93.62 ± 24.00 vs 9.56 ± 1.99, 25.13 ± 16.90 or 12.93 ± 6.14, P < 0.01) and AST (82.32 ± 26.92 vs 11.33 ± 4.17, 18.01 ± 10.00 or 14.00 ± 6.19, P < 0.01) levels in HL+I/R group were observed suggesting severe liver damage, which was confirmed by liver histology. In addition, HL combined with I/R also caused significantly increased hepatocyte apoptosis, as evidenced by increased TUNEL-positive cells (6.20 ± 0.29 vs 1.5 ± 0.22, 4.47 ± 0.45 or 1.97 ± 0.47, P < 0.01), elevated expression of caspase-3 and lower expression of bcl-2. Furthermore, when compared to HL or I/R alone, HL plus I/R enhanced serum TNF-α, IL-1, liver mitochondrial MDA and Ca(2+) levels, suppressed SOD and GSH-Px in liver mitochondria, and markedly up-regulated the activity (11.76 ± 2.36 vs 4.77 ± 2.31 or 3.11 ± 1.35, P < 0.01) and expression (3.24 ± 0.38 vs 1.98 ± 0.88 or 1.72 ± 0.58, P < 0.01) of CYP2E1 in liver. CONCLUSION: The coexistence of HL and acute cerebral I/R induces severe liver damage, suggesting that cerebral ischemic stroke would exaggerate the damage of liver caused by HL. This effect is possibly due to enhanced CYP2E1 induction which further promotes oxidative damage, inflammation and hepatocyte apoptosis.

Optimization of ellagic acid production from ellagitannins by co-culture and correlation between its yield and activities of relevant enzymes.

Aspergillus oryzae was co-cultured with Trichoderma reesei using acorn cups extract containing up to 62% ellagitannins as substrate to produce ellagic acid with relatively high levels of ellagitannin acyl hydrolase, cellulase and xylanase. Ellagitannins concentration, initial pH, T. reesei and A. oryzae during the fermentation were identified as important process parameters effecting ellagic acid accumulation and the enzymes syntheses. These parameters were optimized by uniformity design to determine the optimum condition for ellagic acid production. Under optimum operational condition, ellagic acid yield could be arrived at 24%, when the fermentation run lasted 96h with an initial pH of 4.5, an ellagitannins concentration of 4gl(-1), T. reesei of 3ml and A. oryzae of 3ml. Meanwhile, it was found that the three enzymes activities correlated very well with ellagic acid yield, resulting in model with high coefficient of determination (R(2)=0.98). The results indicate that the mixed culture of T. reesei and A. oryzae is an effective approach to produce an enzyme system of degrading ellagitannins for ellagic acid production.