The synergism of Clinacanthus nutans Lindau extracts with gemcitabine: downregulation of anti-apoptotic markers in squamous pancreatic ductal adenocarcinoma

Background: Clinacanthus nutans extracts have been consumed by the cancer patients with the hope that the extracts can kill cancers more effectively than conventional chemotherapies. Our previous study reported its anti-inflammatory effects were caused by inhibiting Toll-like Receptor-4 (TLR-4) activation. However, we are unsure of its anticancer effect, and its interaction with existing chemotherapy. Methods: We investigated the anti-proliferative efficacy of polar leaf extracts (LP), non-polar leaf extracts (LN), polar stem extract (SP) and non-polar stem extracts (SN) in human breast, colorectal, lung, endometrial, nasopharyngeal, and pancreatic cancer cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT assay. The most potent extracts was tested along with gemcitabine using our established drug combination analysis. The effect of the combinatory treatment in apoptosis were quantified using enzyme-linked immunosorbent assay (ELISA), Annexin V assay, antibody array and immunoblotting. Statistical significance was analysed using one-way analysis of variance (ANOVA) and post hoc Dunnett's test. A p-value of less than 0.05 (p Results: All extracts tested were not able to induce potent anti-proliferative effects. However, it was found that pancreatic ductal adenocarcinoma, PDAC (AsPC1, BxPC3 and SW1990) were the cell lines most sensitive cell lines to SN extracts. This is the first report of C. nutans SN extracts acting in synergy with gemcitabine, the first line chemotherapy for pancreatic cancer, as compared to conventional monotherapy. In the presence of SN extracts, we can reduce the dose of gemcitabine 2.38-5.28 folds but still maintain the effects of gemcitabine in PDAC. SN extracts potentiated the killing of gemcitabine in PDAC by apoptosis. Bax was upregulated while bcl-2, cIAP-2, and XIAP levels were downregulated in SW1990 and BxPC3 cells treated with gemcitabine and SN extracts. The synergism was independent of TLR-4 expression in pancreatic cancer cells. Conclusion: These results provide strong evidence of C. nutans extracts being inefficacious as monotherapy for cancer. Hence, it should not be used as a total substitution for any chemotherapy agents. However, SN extracts may synergise with gemcitabine in the anti-tumor mechanism.</p

A Bis-benzopyrroloisoquinoline Alkaloid Incorporating a Cyclobutane Core and a Chlorophenanthroindolizidine Alkaloid with Cytotoxic Activity from <i>Ficus fistulosa</i> var. <i>tengerensis</i>

Tengerensine (<b>1</b>), isolated as a racemate and constituted from a pair of bis-benzopyrroloisoquinoline enantiomers, and tengechlorenine (<b>2</b>), purified as a scalemic mixture and constituted from a pair of chlorinated phenanthroindolizidine enantiomers, were isolated from the leaves of <i>Ficus fistulosa</i> var. <i>tengerensis</i>, along with three other known alkaloids. The structures of <b>1</b> and <b>2</b> were determined by spectroscopic data interpretation and X-ray diffraction analysis. The enantiomers of <b>1</b> were separated by chiral-phase HPLC, and the absolute configurations of (+)-<b>1</b> and (−)-<b>1</b> were established via experimental and calculated ECD data. Compound <b>1</b> is notable in being a rare unsymmetrical cyclobutane adduct and is the first example of a dimeric benzopyrroloisoquinoline alkaloid, while compound <b>2</b> represents the first naturally occurring halogenated phenanthroindolizidine alkaloid. Compound (+)-<b>1</b> displayed a selective in vitro cytotoxic effect against MDA-MB-468 cells (IC₅₀ 7.4 μM), while compound <b>2</b> showed pronounced in vitro cytotoxic activity against all three breast cancer cell lines tested (MDA-MB-468, MDA-MB-231, and MCF7; IC₅₀ values of 0.038–0.91 μM)

Top 20 pharmaceutical perturbagens exhibiting positive correlation to the gene signature induced by Cud C treatment.

<p>Top 20 pharmaceutical perturbagens exhibiting positive correlation to the gene signature induced by Cud C treatment.</p

Cudraflavone C inhibits PI3K activity.

<p>The effect of negative control (1%DMSO), Cud C or LY-294002 (100 μM) on p110α/p85α, p110β/p85α, p110δ/p85α, and p120γ PI3K activity were quantified using the PI3K-Glo^™ Class I Profiling Kit. All data represents the mean ± s.d. from at least three independent experiments. Symbol “*” presents the statistical significance concluded from Student’s independent <i>t</i>-test with p-value ≤0.05.</p

Cudraflavone C induces tumor-specific cell death in colorectal cancer cells.

<p>(A) Chemical structure of Cud C. (B) KM12, HT29, Caco-2, HCC2998, HCT116 and SW48 colorectal cancer cells were exposed to various concentrations of Cud C for 72 hours. Cell viability was recorded using CellTitre Glo^® luminescence assay. (C) KM12, Caco-2 and CCD 841 CoN were treated with 0.1% DMSO (control) or 10μM Cud C (Cud C) for 72 hours followed by microscopy analysis (×100 magnification). (D) Apoptotic cell death in KM12, Caco-2 and CCD 841 CoN cells was quantified using Annexin V/7-AAD flow cytometry at 72 hours following treatment. (E) Caspase activities in KM12 and Caco-2 cells were assessed by Caspase Glo^® assay at 72 hours following treatment. (F) 10μM Cud C induced mitochondrial membrane depolarization. Caco-2 and KM12 cells stained with JC-1 at 72 hours after treatment with Cud C. The green dye represents JC-1 monomers in cytoplasm while the red dye represents JC-1 aggregates in nucleus. Cells were observed under fluorescence microscope (×100 magnification). All data represent the mean ± s.d. from at least three independent experiments. Symbol “*” presents the statistical significance concluded from Student’s independent <i>t</i>-test with p-value ≤0.05.</p

IC₅₀ of Cud C and 5-fluorouracil in colorectal cancer and non-transformed colon epithelial cells.

<p>IC₅₀ of Cud C and 5-fluorouracil in colorectal cancer and non-transformed colon epithelial cells.</p

Differential gene expression regulated by cudraflavone C in Caco-2 cells.

<p>(A) Heatmaps generated based on the genes regulated by Cud C. Caco-2 cells were exposed to 10 μM Cud C for 48 hours. GeneChip^® Human Transcriptome Array 2.0 (Affymetrix, USA) was applied. Gene expression changes that ≥2-fold were considered significant. Control 1 and Control 2 represent gene expression from cells treated with vehicle control (1% DMSO); Cud C 1 and Cud C 2 were gene expression from cells treated with Cud C (10 μM). (B) qPCR was used to validate the microarray data. Caco-2 cells were exposed to 10 μM Cud C for 12, 24, 48 or 72 hours The left and right panels present genes that are up and down-regulated respectively. All data represent the mean ± s.d. from at least three independent experiments. Symbol “*” indicates the statistical significance concluded from Student’s independent <i>t</i>-test with p-value ≤0.05.</p