GC-MS Method for Quantification and Pharmacokinetic Study of Four Volatile Compounds in Rat Plasma after Oral Administration of Commiphora myrrh (Nees) Engl. Resin and In Vitro Cytotoxic Evaluation

A rapid, simple, and sensitive gas chromatography–tandem mass spectrometry (GC–MS) method was established and validated for simultaneous determination of four volatile compounds, namely curzerene, methoxyfuranodiene, β-elemene, and α-pinene in rat plasma samples after oral administration of the resin extract of Commiphora myrrh using limonene as an internal standard (IS). Liquid-liquid extraction using hexane and ethyl acetate (1:1) mixture as an extracting agent was used for the samples extraction procedure. The GC–MS system was operated under selective ion monitoring (SIM) mode using Perkin Elmer Elite 5MS column (30 m × 0.25 mm × 0.25 µm film thickness). Specificity, linearity, precision, accuracy, extraction recovery, and stability were used to validate the developed method. The assay showed good linearity (r2 ≥ 0.998), and the lowest limits of quantification (LLOQ) were 3.97–21.38 ng/mL for the four analytes. This assay was successfully applied to pharmacokinetic studies of the four volatile compounds in rat plasma. The antiproliferative activity of these volatile compounds was evaluated against lung carcinoma (A549) and colon (LoVo) cell lines, were each compound caused variable inhibition on cells proliferation and methoxyfuranodiene exerted the strong antiproliferative activity against both cell line according to IC50 values.

Exosomal lncRNA PVT1/VEGFA Axis Promotes Colon Cancer Metastasis and Stemness by Downregulation of Tumor Suppressor miR-152-3p

Background. Treating advanced colon cancer remains challenging in clinical settings because of the development of drug resistance and distant metastasis. Mechanisms underlying the metastasis of colon cancer are complex and unclear. Methods. Computational analysis was performed to determine genes associated with the exosomal long noncoding (lncRNA) plasmacytoma variant translocation 1 (PVT1)/vascular endothelial growth factor A (VEGFA) axis in patients with colon cancer. The biological importance of the exosomal lncRNA PVT1/VEGFA axis was examined in vitro by using HCT116 and LoVo cell lines and in vivo by using a patient-derived xenograft (PDX) mouse model through knockdown (by silencing of PVT1) and overexpression (by adding serum exosomes isolated from patients with distant metastasis (M-exo)). Results. The in silico analysis demonstrated that PVT1 overexpression was associated with poor prognosis and increased expression of metastatic markers such as VEGFA and epidermal growth factor receptor (EGFR). This finding was further validated in a small cohort of patients with colon cancer in whom increased PVT1 expression was correlated with colon cancer incidence, disease recurrence, and distant metastasis. M-exo were enriched with PVT1 and VEGFA, and both migratory and invasive abilities of colon cancer cell lines increased when they were cocultured with M-exo. The metastasis-promoting effect was accompanied by increased expression of Twist1, vimentin, and MMP2. M-exo promoted metastasis in PDX mice. In vitro silencing of PVT1 reduced colon tumorigenic properties including migratory, invasive, colony forming, and tumorsphere generation abilities. Further analysis revealed that PVT1, VEGFA, and EGFR interact with and are regulated by miR-152-3p. Increased miR-152-3p expression reduced tumorigenesis, where increased tumorigenesis was observed when miR-152-3p expression was downregulated. Conclusion. Exosomal PVT1 promotes colon cancer metastasis through its association with EGFR and VEGFA expression. miR-152-3p targets both PVT1 and VEGFA, and this regulatory pathway can be explored for drug development and as a prognostic biomarker.

Rauwolfia vomitoria Extract Represses Colorectal Cancer Cell Autophagy and Promotes Apoptosis

<b><i>Background:</i></b> Colorectal cancer (CRC) is one of the most frequent digestive tract tumors in the world with an increasing incidence. Currently, surgical resection and chemotherapy are the main therapeutic options; however, their effects are limited by various adverse reactions. <i>Rauwolfia vomitoria</i> extract (Rau) has been shown to repress the progression of multiple human cancers; however, whether Rau plays a role in CRC remains undetermined. <b><i>Methods:</i></b> Influences of Rau treatment on HCT-116 and LoVo cells were estimated via MTT and colony formation experiments. Flow cytometry analysis was adopted to evaluate the apoptosis rate of HCT-116 and LoVo cells. Apoptosis-related proteins (Bcl-2, Bax, and caspase-3) and autophagy-related proteins (LC3 and P62) were assessed by Western blotting. Effects of Rau on autophagy of HCT-116 and LoVo cell were evaluated through GFP-LC3 analysis. In vivo xenograft tumor assay was conducted to further examine the role of Rau in CRC tumor growth. <b><i>Results:</i></b> Rau remarkably repressed HCT-116 and LoVo cell viability and promoted HCT-116 and LoVo cell apoptosis in vitro in a dose-dependent manner. Rau increased the expression of caspase-3 and Bax and decreased the expression of Bcl-2 in HCT-116 and LoVo cells. Moreover, Rau was demonstrated to decrease the LC3||/LC3| ratio and increase the level of P62 in HCT-116 and LoVo cells. In addition, we found that Rau repressed xenograft tumor growth and also repressed autophagy in vivo. <b><i>Conclusion:</i></b> Our findings revealed that Rau repressed CRC cell viability and autophagy in vitro and in vivo, suggesting that Rau might be a potent therapeutic agent of CRC.

Exosomal lncRNA PVT1/VEGFA Axis Promotes Colon Cancer Metastasis and Stemness by Downregulation of Tumor Suppressor miR-152-3p

Background: Late-stage colon cancer remains a treatment challenge in clinical settings because of the development of drug resistance and distant metastasis. Nevertheless, the mechanisms through which colon cancer cells acquire the ability to metastasize are complicated and require more research.Methods: Bioinformatic analysis was performed to determine gene associated with exosomal lncRNA PVT1/VEGFA axis of colon cancer patients. Biological importance of exosomal lncRNA PVT1/VEGFA axis was investigated in vitro (HCT116 and LoVo cell lines) and in vivo (PDX mouse model) through knockdown (siPVT1) and overexpression (add exosomes from sera of distant metastasis patients). PVT1/VEGFA axis related protein expression in and cell lines were investigated through RT-qPCR, immunoblotting, and immunohistochemistry analysis. Colony formation Assay, cell invasion, migration, and tumorsphere-formation assay were used to explore possible molecular mechanism. Results: First, using public databases, we demonstrated that PVT1 overexpression is associated with poor prognosis and increased metastatic markers, such as vascular endothelial growth factor A (VEGFA) and epidermal growth factor receptor (EGFR). This finding was then validated in a small cohort of patients with colon cancer, where increased PVT1 expression was correlated with colon cancer incidence, disease recurrence, and distant metastasis. Notably, serum exosomes from patients with metastatic (M-exo) colon cancer were enriched with PVT1 and VEGFA and increased both migratory and invasive abilities in colon cancer cell lines when cocultured. This metastasis-promoting effect was accompanied by an increased expression of Twist1, Vimentin, and MMP2. Notably, M-exo promoted metastatic incidence in patient-derived xenograft mice. In vitro silencing of PVT1 led to decreased colon tumorigenic properties, including colony formation, tumorsphere formation, and metastatic potential. Further analysis revealed that miR-152-3p has multiple targets, including PVT1, VEGFA, and EGFR. Increased miR-152-3p resulted in decreased tumorigenesis, and the reverse was true when the miR-152-3p level was decreased.Conclusion: In conclusion, we provided evidence regarding the role of exosomal PVT1 in promoting metastasis in colon cancer through its association with EGFR and VEGFA expression. PVT1 and VEGFA are both targets of miR-152-3p, and this regulatory pathway could be explored for drug and prognostic biomarker development.

ALKBH2 inhibition alleviates malignancy in colorectal cancer by regulating BMI1-mediated activation of NF-κB pathway

Background The alkB homolog 2, alpha-ketoglutarate-dependent dioxygenase (ALKBH2) gene is involved in DNA repair and is expressed in different types of malignancies. However, the role of ALKBH2 in colorectal carcinoma (CRC) remains unclear. This study aimed to explore the potential mechanism of ALKBH2 and its function in CRC. Methods The expression levels of ALKBH2 in CRC tissues and cells were determined by qRT-PCR. Following that, the role of ALKBH2 in cell proliferation, invasion, and epithelial-mesenchymal transition (EMT) in CRC cells (Caco-2 and LOVO) were assessed by Cell Counting Kit-8 (CCK-8), transwell assays, and Western blotting, respectively. The effect of ALKBH2 on B cell-specific Moloney murine leukemia virus integration site 1 (BMI1) and downstream NF-κB pathway was determined by Western blotting and luciferase reporter assay. Results The expression of ALKBH2 was significantly upregulated both in CRC tissues and cells. Further experiments demonstrated that reduction of ALKBH2 suppressed Caco-2 and LOVO cell proliferation and invasion. Moreover, ALKBH2 knockdown also suppressed EMT, which increased E-cadherin expression and reduced N-cadherin expression. Besides, ALKBH2 silencing inhibited BMI1 expression and reduced nuclear accumulation of the NF-κB p65 protein, as well as the luciferase activity of NF-κB p65. Upregulation of BMI1 reversed the effect of ALKBH2 knockdown on the proliferation and invasion in CRC cells. Conclusions Our findings suggest that suppression of ALKBH2 alleviates malignancy in CRC by regulating BMI1-mediated activation of NF-κB pathway. ALKBH2 may serve as a potential treatment target for human CRC.

CCDC12 Promotes Tumor Development and Invasion Through the Snail Pathway in Colon Adenocarcinoma

Background: To determine the role of Coiled-coil domain containing 12 (CCDC12) in colon adenocarcinoma (COAD).Methods: We used integrative expression Quantitative Trait Loci (eQTL) analysis to identify risk single nucleotide polymorphisms (SNPs) and associated genes. Immunohistochemical staining (IHC) and western blotting (WB) were used to detect CCDC12 expression. We selected SW480 and LOVO cell lines to knock down CCDC12, while HCT116 and SW480-KD cell lines were up-regulated. Then performed functional studies and established a xenograft tumor model in BALB/c mice. Four plex Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) assays were performed to determine its function and potential regulatory mechanism. Overexpressed Snail and knocked down CCDC12 subsequently in SW480 cells to find them association.Results: Integrative eQTL analysis found that rs8180040 was significantly associated with CCDC12 in COAD patients. IHC and WB confirmed CCDC12 was highly expressed in COAD tissues (IHC 51/75 vs 8/75, WB 10/12 vs 2/12). This was consistent with RNA-Seq data from TCGA database (P value < 0.001). Knockdown of CCDC12 could significantly reduce proliferation, migration, invasion and tumorigenicity of colon cancer cells, while exogenous overexpression of CCDC12 had the opposite effect. iTRAQ assays demonstrated that overexpression of CCDC12 would change proteins on adherens junction pathway. Overexpression of Snail did not significantly change CCDC12 levels in SW480 cells, while knock down of CCDC12 reduced that of Snail.Conclusions: CCDC12 plays a significant role in tumorigenesis, development and invasion of COAD and may affect the epithelial to mesenchymal transformation process of colon cancer cells by regulating the Snail pathway.

Podoplanin-mediated platelet activation promotes proliferation and invasion of colon cancer cells

Background: Recent studies have shown that podoplanin is highly expressed in many tumors, suggesting that podoplanin may be related to the invasion and metastasis of malignant tumors. A potential mechanism by which podoplanin promote tumor invasion and metastasis is podoplanin-mediated platelet aggregation and activation.Methods: The expression of podoplanin in colon cancer LOVO cell line and lung adenocarcinoma cell line A549 was detected by immunoblotting. The above cells were co-cultured with washed mouse platelets. The platelet aggregation meter was used to determine the platelet aggregation rate and observe the cell morphology. Immunofluorescence staining and immunoblotting were used to detect the expression of EMT-related factors and the phosphorylation level of Smad2/3 downstream of the TGF-β signal; and then the matrigel-coated transwell chamber invasion test was used to evaluate the invasion ability of tumor cells. Lentiviruses targeting human podoplanin shRNA were constructed to infect colon cancer LOVO cells to generate cell lines with stable podoplanin knockdown, and the above experiments were repeated. Results: The colon cancer LOVO cell line with positive podoplanin expression had platelet aggregation activity, and the platelet aggregation activity of colon cancer LOVO cell line disappeared when anti-podoplanin monoclonal antibody MS-140 was added. However, no podoplanin expression and platelet aggregation activity were detected in lung adenocarcinoma cell line A549. When colon cancer LOVO cell-platelet reactant supernatant was added to colon cancer LOVO cell culture, the morphology of colon cancer LOVO cells showed EMT characteristics. Immunoblot showed that E-cadherin was down-regulated in colon cancer LOVO cells, while Vimentin And N-cadherin expression are upregulated. However, in lung adenocarcinoma A549 cells, there were no changes in EMT morphology and EMT factors. Immunoblot and ELISA showed that colon cancer LOVO cells with positive expression of podoplanin released TGF-β after aggregating with platelets, activated the TGF-β/Smad2/3 signaling pathway, and induced EMT and increased invasiveness of colon cancer LOVO cells. Treating colon cancer LOVO cells with TGF-β antibodies and TGF-β receptor inhibitors, or using LOVO cell lines with podoplanin knockdown, and repeating the above experiments suggest that platelet aggregation activity disappears and tumor cells EMT are eliminated, and The ability to invade is reduced. Conclusions: Podoplanin-mediated platelet activation plays an important role in colon cancer invasion and metastasis; its possible mechanism is that colon cancer LOVO cells with positive podoplanin expression react with platelets, activate platelets to release TGF-β, and activate TGF-β / Smad2 / 3 Signaling pathway induces EMT in colon cancer LOVO cells and enhances tumor cell proliferation and invasiveness.

Novel Polymethoxylated Chalcones as Potential Compounds Against KRAS-Mutant Colorectal Cancers

Background/Objective: KRAS-mutant colorectal cancers (CRC) are tumors that are associated with poor prognosis. However, no effective treatments are available to target them. Therefore, we designed and synthesized novel chalcone analogs, small organic molecules, to investigate their effects on KRAS-mutant CRC cells. Methods: Fourteen new chalcone analogs were synthesized, optimized, characterized, and tested against two KRAS-mutant CRC cell lines (HCT-116 and LoVo), one p-53 and BRAF mutant CRC cell line (HT-29) and one normal immortalized colon cells (NCE-1 E6/E7). Effects on cell viability, apoptosis, cell cycle, migration, colony formation, EMT, and angiogenesis were investigated. Results: Compounds 3 and 14 were the most effective. Compound 3 showed potent activity against HCT-116 and LoVo cell lines (GI50 of 6.10 μM and 7.00 μM, respectively). While compound 14 showed GI50 of 8.60 μM and 8.80 μM on HCT-116 and LoVo cell lines, respectively. Both compounds were approximately 2-3 times more selective toward cancer cells rather than normal colon cells. Compound 3 was effective in inducing apoptosis in HCT-116 cells via Bax upregulation and Bcl-2 downregulation. Invasion and metastasis of KRAS-mutant cells were modulated by compounds 3 and 14 through significant inhibition of cell migration and the prevention of colony formation. In addition, they reversed EMT by downregulation of EMT markers (vimentin, fascin, and β- catenin) and upregulation of cell-cell adhesion marker, E-cadherin. Furthermore, compounds 3 and 14 had significantly inhibited angiogenesis in ovo. Conclusion: Compounds 3 and 14 represent potent and selective leads for KRAS-mutant CRC cells, thus, further in vitro and in vivo studies are necessary to confirm their effect on KRAS-mutant CRCs.

Tenacissoside H Induces Apoptosis and Inhibits Migration of Colon Cancer Cells by Downregulating Expression of GOLPH3 Gene

Objective. Tenacissoside H (TDH) is a Chinese medicine monomer extracted from Marsdenia tenacissima extract (MTE), which has been confirmed to have antitumor effects, but its mechanism is still unclear. The aim of this study was to investigate the effect and mechanism of TDH on human colon cancer LoVo cell proliferation and migration and explore the correlation of TDH treatment with the expression of GOLPH3 and cell signaling pathways in LoVo cells. Methods. LoVo cells were treated with TDH at 0.1, 1, 10, and 100 μg/mL for 24, 48, and 72 h. The proliferation rate of LoVo cells was evaluated by MTT assay. Recombinant plasmid p-CMV-2-GOLPH3 was constructed, and p-CMV-2-GOLPH3 and p-CMV-2 empty plasmids were transfected into LoVo cells by lipofection. Western blotting was used to detect the transfection efficiency and the expression of p-p70S6K, p70S6K, β-catenin, and GOLPH3. The apoptosis rate was analyzed with Annexin V-FITC/PI double-staining method, and cell migration assessed by transwell assay. Results. TDH inhibited the proliferation of LoVo cells in a concentration-dependent manner. The IC50 of TDH treatment in LoVo cells at 24, 48, and 72 h was 40.24, 13.00, and 5.73 μg/mL, respectively. TDH treatment significantly induced apoptosis and suppressed the viability and migration of human colon cancer LoVo cells. The effect of TDH on induction of apoptosis and inhibition of migration in LoVo cells decreased significantly after activating the PI3K/AKT/mTOR and Wnt/β-catenin signaling pathways with agonists. Additionally, the expression of GOLPH3 protein downregulated significantly in LoVo cells under TDH treatment. Overexpression of the GOLPH3 gene increased the expression of key proteins in PI3K/AKT/mTOR and Wnt/β-catenin signaling pathways and blocked the antitumor activity of TDH. Conclusion. Collectively, the present results indicated that TDH can inhibit the proliferation vitality of colon cancer LoVo cells through downregulating GOLPH3 expression and activity of PI3K/AKT/mTOR and Wnt/β-catenin signaling pathways.

The Effect of 4′-hydroxy-3,4,5-trimetoxystilbene, the Metabolite of Resveratrol Analogue DMU-212, on Growth, Cell Cycle and Apoptosis in DLD-1 and LOVO Colon Cancer Cell Lines

Resveratrol is a phytoalexin that naturally occurs in grapes, blueberries, cranberries, peanuts and many other plants. Although resveratrol inhibits carcinogenesis in all three stages, its clinical application is restricted due to poor pharmacokinetics. The methylated analogues of resveratrol have been found to have higher bioavailability and cytotoxic activity than that of the prototupe compound. Among the various methoxy derivatives of resveratrol, 3,4,5,4′-tetrametoxystilbene (DMU-212) is suggested to be one of the strongest activators of cytotoxicity and apoptosis. DMU-212 has been shown to exert anti-tumor activity in DLD-1 and LOVO colon cancer cells. Since colorectal cancer is the third most common cause of cancer-related deaths worldwide, the development of new anticancer agents is nowadays of high significance. The aim of the present study was to assess the anticancer activity of 4′-hydroxy-3,4,5-trimetoxystilbene (DMU-281), the metabolite of DMU-212, in DLD-1 and LOVO cell lines. We showed for the first time the cytotoxic activity of DMU-281 triggered via cell cycle arrest at G2/M phase and apoptosis induction accompanied by the activation of caspases-9, -8, -3/7. Furthermore, DMU-281 has been found to change the expression pattern of genes and proteins related to intrinsic as well as extrinsic apoptosis. Since the activation of these pathways of apoptosis is still the most desired strategy in anticancer research, DMU-281 seems to provide a promising approach to the treatment of colon cancer.