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Table S5 shows 16 up-regulated genes and 10 predicted potential transcript factors in the global expression analysis

Table S1 shows clinicopathologic characteristics of 50 CRC patients

Figure S2 indicates that higher PNO1 mRNA levels are associated with poorer overall survival, relapse-free survival and shorter time of first progression of patients with breast or lung cancer. PNO1 knockdown decreased cell viability and cell survival of lung and breast cancer cells.

Table S4 summaries the characteristics of antibodies for analyses of Western-bloting, immunofluorescence, immunohistochemistry and co-immunoprecipitation.

Table S6 indicates that PNO1 mRNA expression is significantly up-regulated in various types of malignancies, including CRC, lung cancer, and breast cancer.

The RNA-binding protein PNO1 is critical for ribosome biogenesis, but its potential role in cancer remains unknown. In this study, online data mining, cDNA, and tissue microarrays indicated that PNO1 expression was higher in colorectal cancer tissue than in noncancerous tissue, and its overexpression was associated with worse patient survival. Gain-of-function and loss-of-function studies demonstrated that PNO1 knockdown suppressed growth of colorectal cancer cells in vitro and in vivo, while PNO1 overexpression promoted colorectal cancer cell proliferation in vitro. In colorectal cancer cells expressing wild-type p53, PNO1 knockdown enhanced expression of p53 and its downstream gene p21, and reduced cell viability; these effects were prevented by p53 knockout and attenuated by the p53 inhibitor PFT-α. Moreover, PNO1 knockdown in HCT116 cells decreased levels of 18S rRNA, of 40S and 60S ribosomal subunits, and of the 80S ribosome. It also reduced global protein synthesis, increasing nuclear stress and inhibiting MDM2-mediated ubiquitination and p53 degradation. Overexpressing EBF1 suppressed PNO1 promoter activity and decreased PNO1 mRNA and protein, inhibiting cell proliferation and inducing cell apoptosis through the p53/p21 pathway. In colorectal cancer tissues, the expression of EBF1 correlated inversely with PNO1. Data mining of online breast and lung cancer databases showed increased PNO1 expression and association with poor patient survival; PNO1 knockdown reduced cell viability of cultured breast and lung cancer cells. Taken together, these findings indicate that PNO1 is overexpressed in colorectal cancer and correlates with poor patient survival, and that PNO1 exerts oncogenic effects, at least, in part, by altering ribosome biogenesis.Significance:This study identifies the ribosome assembly factor PNO1 as a potential oncogene involved in tumor growth and progression of colorectal cancer.

Figure S1 shows that the expression levels of endogenous PNO1 in cultured CRC cell lines are relatively higher than that of differentiated Caco2 cells, and PNO1 knockdown reduces cell viability and survival of CRC cells.

lncRNA ANRIL is an oncogene, however the role of ANRIL in the regulation of colorectal cancer on human lymphatic endothelial cells (HLECs) is remain elusive. Pien Tze Huang (PZH, PTH) a Tradition Chinese Medicine (TCM) as an adjunctive medication could inhibit the cancer metastasis, however the mechanism still uncovering. We used network pharmacology, subcutaneous and orthotopic transplanted colorectal tumors models to determine the effect of PZH on tumor metastasis. Differential expressions of ANRIL in colorectal cancer cells, and stimulating the regulation of cancer cells on HLECs by culturing HLECs with cancer cells’ supernatants. Network pharmacology, transcriptomics, and rescue experiments were carried out to verify key targets of PZH. We found PZH interfered with 32.2% of disease genes and 76.7% of pathways, and inhibited the growth of colorectal tumors, liver metastasis, and the expression of ANRIL. The overexpression of ANRIL promoted the regulation of cancer cells on HLECs, leading to lymphangiogenesis, via upregulated VEGF-C secretion, and alleviated the effect of PZH on inhibiting the regulation of cancer cells on HLECs. Transcriptomic, network pharmacology and rescue experiments show that PI3K/AKT pathway is the most important pathway for PZH to affect tumor metastasis via ANRIL. In conclusion, PZH inhibits the regulation of colorectal cancer on HLECs to alleviate tumor lymphangiogenesis and metastasis by downregulating ANRIL dependent PI3K/AKT/VEGF-C pathway.

Background: Anlotinib is a multi-target tyrosine kinase inhibitor that has been reported to have activity against colorectal cancer. However, the mechanisms of how anlotinib mediates drug-resistance of colorectal cancer have not been fully described. Particularly the potential mechanisms regarding the inhibition of proliferation and induction of apoptosis remain unknown. Objective: In this study, we intended to study the effect and related-mechanism of the proliferation, migration, invasion and induced apoptosis of anlotinib overcoming multidrug resistant colorectal cancer cells through in vitro experiments. Methods: Cell viability was determined by MTT assays and the resistant index was calculated. Colony formation and PI/RNase Staining were used for testing the proliferation of resistant cells. DAPI staining and Annexin V-FITC/PI staining were used to detect cell apoptosis. Migration and invasion were examined by transwell. Protein expression and activation of PI3K/AKT pathway were detected by western blot. LY294002 was used to verify whether anlotinib overcomes the drug-resistance of CRC cells by inactivating the PI3K/AKT pathway. Results: The results showed that the HCT-8/5-FU cells were resistant to multiple chemotherapy drugs (5-FU, ADM and DDP). Anlotinib significantly inhibited cell viability, proliferation, migration, invasion and induced cell apoptosis. Moreover, anlotinib down-regulated the expression of survivin, cyclin D1, CDK4, caspase-3, Bcl-2, MMP-2, MMP-9, vimentin and N-cadherin, but up-regulated cleaved-caspase-3, Bax and E-cadherin and blocked the activity of the PI3K/AKT in HCT-8/5-FU cells. We found anlotinib and LY294002 overcame the drug resistance of HCT-8/5-FU cells by reducing the expression of PI3K/p-AKT. Conclusions: Anlotinib inhibited the proliferation, migration, invasion and induced apoptosis of HCT-8/5-FU cells, and the mechanisms may be that anlotinib conquered multidrug resistance of colorectal cancer cells via inactivating of PI3K/AKT pathway.

Multidrug resistance (MDR) is a critical reason for cancer chemotherapy failure. Babaodan (BBD) is a famous traditional Chinese patent medicine reported to have antigastric cancer activity. However, the roles and molecular mechanisms of the reversal of MDR of gastric cancer by BBD have not been well described until now. Therefore, the purpose of this study was to elucidate further the role of BBD in reversing the MDR of gastric cancer cells and its specific regulatory mechanism via in vitro experiments. To verify our results, MTT, Doxorubicin (DOX) staining, Rhodamin123 (Rho123) staining, DAPI staining, Annexin V-FITC, propidium iodide (PI), Cyto-ID, and western blot assays were performed. To determine whether BBD triggers apoptosis and autophagy through the PI3K/AKT/mTOR signaling, we also applied 3-methyladenine (3-MA), chloroquine (CQ), and 740Y-P (an activator of PI3K). The results showed that BBD reversed the MDR and induced apoptosis and autophagy of SGC7901/DDP cells. Pathway analyses suggested BBD inhibits PI3K/AKT/mTOR pathway activity and subsequent apoptosis-autophagy induction. Inhibition of autophagy with 3-MA and chloroquine (CQ) was performed to confirm that BBD promoted autophagy. PI3K agonist, 740Y-P, further verified BBD inhibition of PI3K/AKT/mTOR pathway activation. In conclusion, BBD may reverse the MDR of gastric cancer cells, induce apoptosis, and promote autophagy via inactivation of the PI3K/AKT/mTOR signaling pathway.

Colorectal cancer (CRC), mostly categorized as a low immunogenic microsatellite-stable phenotype bearing complex immunosuppressive tumor microenvironment (TME), is highly resistant to immunotherapy. Seeking safe and efficient alternatives aimed at modulating tumor immunosuppressive TME to improve outcome of CRC is highly anticipated yet remains challenging.

Background To further elucidate the anti-angiogenesis effect of Babao Dan (BBD) in vitro, gastric cancer (GC) cells and human umbilical vein endothelial cells (HUVECs) were used to evaluate the regulation role of BBD by vascular endothelial growth factor A (VEGFA)/vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathway. Methods After induced by VEGFA, GC cells (AGS, MGC80-3 and BGC823) were treated by different concentrations of BBD and then were detected cell viability, migration and VEGFA level. And the anti-angiogenesis effect of BBD was evaluated with HUVECs. To furtherly mimic the tumor microenvironment of angiogenesis, VEGFA as an inducer (10 ng/mL) was used to trigger a cascade of angiogenesis of HUVECs in vitro. Results The viability and migration of GC cells with VEGFA-induced or non-induced and VEGFA levels in GC cells were significantly inhibited by BBD with concentration-dependent manner (P

Background Sonic Hedgehog (SHh) signaling pathway plays a critical role in cell proliferation, apoptosis, and tumor angiogenesis in various types of malignancies including colorectal cancer (CRC). Qingjie Fuzheng Granules (QFG) is a traditional Chinese medicinal formula, which has been clinically used in various cancer treatments, including CRC. In this study, we explored the potential molecular mechanisms of QFG treatment effects on CRC via the SHh pathway. Methods A CRC HCT-116 xenograft mouse model was utilized for all experiments. Mice were treated with intra-gastric administration of 1 g/kg of QFG or saline 6 days a week for 28 days (4 weeks). Body weight, length and shortest diameter of the tumor were measured every 3 days. At the end of the treatment, the tumor weight was measured. TUNEL staining assays were used to detect tumor apoptosis. Western blot and immunohistochemistry (IHC) assays were used to detect the expression of relative proteins. Results In our results, QFG inhibited the increase of tumor volume and weight, and exhibited no impact on mouse body weight. Furthermore, QFG significantly decreased the expression of SHh, Smo and Gli proteins, indicating the action of SHh signaling. Consequently, the expression of pro-proliferative survivin, Ki-67, Cyclin-D1 and CDK4 were decreased and expression of anti-proliferative p21 was increased. The pro-apoptotic Bax/Bcl-2 ratio, cle-caspase-3 and TUNEL-positive cell percentage in tumor tissues were increased. Meanwhile, the pro-angiogenic VEGF-A and VEGFR-2 expression was down-regulated. Conclusions QFG inhibited CRC cell proliferation and promoted CRC cell apoptosis and tumor angiogenesis in vivo through the suppression of SHh pathway, suggesting that QFG could be a potential therapeutic drug for CRC.

Mini‐chromosome maintenance (MCM) proteins play important roles in initiating eukaryotic genome replication. The MCM family of proteins includes several members associated with the development and progression of certain cancers. We performed online data mining to assess the expression of MCMs in gastric cancer (GC) and the correlation between their expression and survival in patients with GC. Notably, MCM8 expression was undoubtedly up‐regulated in GC, and higher expression correlated with shorter overall survival (OS) and progression‐free survival (PFS) in patients with GC. However, the role of MCM8 in GC has not been previously explored. Our in vitro experiments revealed that MCM8 knockdown inhibited cell growth and metastasis. Moreover, MCM8 knockdown induced apoptosis. Mechanistically, the expression levels of Bax and cleaved caspase‐3 were increased, whereas Bcl‐2 expression decreased. Additionally, we demonstrated that MCM8 knockdown suppressed tumorigenesis in vivo. Overall, these results suggest that MCM8 plays a significant role in GC progression.

Background Liver metastasis is a common cause of death from colorectal cancer (CRC). In this paper we developed a liver metastasis mouse model by microsurgical orthotopic implantation (MSOI) to illuminate the CRC progression with an eye toward developing effective drug treatment. Methods Murine colon carcinoma CT-26 cells were cultured and then injected to male BALB/c athymic nude mice right flank to generate subcutaneous implantation tumor with 2×107 CT-26 cell suspension in DMEM. Tumor tissue at an average size of 1 cm3 was injected into another nude mice right flank with 20-gauge inoculating needle. Between fourth and sixth generations, tumor tissue sewn into the cecal surface establishes orthotopic transplanted CRC model by MSOI. Then on the 7th, 14th, 21st and 35th day, body weight, abdomen circumference, volume of ascites and local tumor weight were observed and weighed. On the 21st day and 35th day, local tumor rate was calculated, and metastatic tumors of other organs were observed. Tumor tissue was stained by HE for pathologic analysis. Results On the 35th day, body weight and abdomen circumference of the model group were significantly higher than the control group (P

Aim: To identify potential key candidate genes, whose expression and clinical significance was further assessed in colorectal cancer (CRC). Methods: Three original microarray datasets (GSE41328, GSE22598, and GSE23878) from NCBI-GEO were used to analyze differentially expressed genes (DEGs) in CRC. Online database analyses through Oncomine and GEIPA were performed to evaluate SLC4A4 expression and explore the prognostic merit of SLC4A4 expression, which was further confirmed by analyses from QPCR based cDNA array and IHC based tissue microarray (TMA). STRING website was used to explore the interaction between SLC4A4 with other DEGs based on the protein-protein interaction (PPI) networks. Results: Analysis of three original microarray datasets from GEO identified 82 shared, differentially expressed genes (28 upregulated and 54 down-regulated) in CRC tissues. Online analyses from Oncomine and GEIPA revealed lower SLC4A4 mRNA expression in CRC tissues compared to adjacent normal tissues, which were further confirmed by QPCR based cDNA array and IHC based TMA analyses on both mRNA and protein levels. Survival analyses through GEIPA and from TMA demonstrated that low SLC4A4 expression is correlated with worse overall survival among patients with CRC. Survival analysis from Kaplan-meier plotter demonstrated that low SLC4A4 expression is significantly associated with poor progression (including relapse-free survival, overall survival, distant metastasis-free survival, post-progression survival) of patients with breast cancer, lung cancer, gastric cancer, and ovarian cancer. PPI analysis found that SLC4A4 is highly correlated with various genes, including SLC9A3, SLC26A6, ENSG00000214921, SLC26A4, SLC9A3R1, and SLC9A1. Conclusion: The mRNA and protein levels of SLC4A4 were decreased in CRC tissues, and low expression of SLC4A4 significantly correlated with shorter survival of CRC patients and poorer progression of patients with breast cancer, lung cancer, gastric cancer and ovarian cancer, suggesting potential role of SLC4A4 on tumor suppression and prognostic prediction in multiple malignancies including CRC.

Qingjie Fuzheng granules (QFG) exert an anticancer effect against colorectal cancers (CRC). However, the pharmacological molecular mechanisms are still unclear. This study was aimed to establish a simple method to predict targets of QFG against CRC by the network pharmacology strategy. 461 compounds and 1559 targets in QFG were enriched by BATMAN-TCM. 21 of the common targets were obtained by the groups of “Jun,” “Chen,” “Zuo,” and “Shi” medicine in QFG. The enrichment analyses of GO functional terms, KEGG pathway, and OMIM/TTD diseases displayed the targets in the different and complementary effects of four functional medicines in QFG. Then, 613 differential targets for QFG in CRC were identified. GO functional terms and KEGG pathway analyses showed that QFG regulated the inflammatory function and lipid metabolic process. There were also targets that played a role in the binding to the receptors in membranes, in the activation of the transportation signal, and provided pain relief by regulation of the neural related pathways. Next, the protein-protein interaction network was analyzed, and the levels of the predicted targets in CRC primary tumor were explored, and 7 candidate targets of QFG against CRC were obtained. Furthermore, with real-time PCR and enzyme-linked immunosorbent assay (ELISA) analysis, downregulation of dopamine D2 receptor (DRD2) and interleukin-6 (IL-6), and upregulation of interleukin-10 (IL-10) were identified following the treatment of QFG. At last, the survival and prognosis of the potential targets of QFG in CRC patients were analyzed by GenomicScape, and IL-6 was suggested to be an index for the regulation of QFG in CRC. These results might elucidate the possible antitumor mechanism of QFG and highlight the candidate therapeutic targets and the application direction in clinical treatment for QFG.

Glypican-2 (GPC2) has been reported to promote tumor progression through metabolic pathways. However, the role of GPC2 in colon adenocarcinoma (COAD) remains to be further investigated. This study was designed to evaluate the role of GPC2 in COAD. Based on patients with complete clinical information and GPC2 expression from the Cancer Genome Atlas-COAD database, we found that GPC2 mRNA was highly expressed in COAD tissues, which was associated with poor prognosis and tumornode-metastasis (TNM) stage. The predicted survival probability based on GPC2 mRNA expression and TNM stage was in good agreement with the observed survival probability. Furthermore, the genes coexpressed with GPC2 in COAD tissues were significantly enriched in basal cell carcinoma, Notch signaling pathway, and Hedgehog signaling pathway. After GPC2 was decreased through transfecting short hairpin RNA of GPC2 into HCT-8 and SW620 cells, cell cycle was arrested in G0/G1 phase, proliferation was decreased, apoptosis was increased, and migration and invasion were repressed. In conclusion, decreasing GPC2 significantly inhibited proliferation, migration, and invasion, and enhanced apoptosis, which implied that GPC2 can be considered a promising therapeutic target of COAD in the future.

Scope To investigate the effect of Qingjie Fuzheng Granule (QFG) on lymphangiogenesis and lymphatic metastasis in colorectal cancer. Methods The effects of QFG on the expression and secretion of vascular endothelial growth factor-C (VEGF-C) in HCT-116 cells were investigated both in vitro and in vivo. HCT-116 cells were treated with different concentrations (0.2, 0.5, and 1.0 mg/mL) of QFG. The VEGF-C expression level was determined using RT-qPCR and western blotting, and the VEGF-C concentration in supernatant was measured by ELISA. Tumor xenograft models of HCT-116 cells were generated using BALB/c nude mice, and the mice were randomly divided into a control group (gavaged with normal saline) and QFG group (gavaged with 2 g/kg QFG). The effect of QFG on tumor growth was evaluated by comparing the volume and weight of tumors between two groups. Immunohistochemistry (IHC) and RT-qPCR were performed to detect the expression levels of VEGF-C, vascular endothelial growth factor receptor 3 (VEGFR-3), and LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1). ELISA was performed to measure the concentration of serum VEGF-C. TMT proteomics technology and Reactome pathway analysis were used to explore the mechanism of QFG inhibiting lymphangiogenesis in tumor. The VEGF-C (5 ng/mL)-stimulated human lymphatic endothelial cell (HLEC) model was conducted to evaluate the effect of QFG on lymphangiogenesis in vitro. The model cells were treated with different concentrations (0.2, 0.5, and 1.0 mg/mL) of QFG. Cell viability was then determined using an MTT assay. The cell migration, invasion, and tube-formation ability were analyzed using transwell migration, matrigel invasion and tube formation assays, respectively. The underlying mechanism was uncovered, the levels of VEGFR-3, matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), p-PI3K/PI3K, p-AKT/AKT and p-mTOR/ mTOR were detected using western blotting. Results QFG significantly reduced VEGF-C expression and secretion in HCT-116 cells. QFG evidently suppressed in vivo tumor growth and the expression of VEGF-C, VEGFR-3, and LYVE-1. The serum VEGF-C level was also reduced by QFG. Moreover, TMT proteomics technology and Reactome pathway analysis identified 95 differentially expressed protein and multiple enriched pathway about matrix metalloproteinase and extracellular matrix, which is direct associate with lymphangiogenesis. In vitro experiment, QFG inhibited the viability, migration, invasion and tube formation of HLECs. Additionally, QFG reduced the VEGFR-3, MMP-2, MMP-9 expression levels, and the p-PI3K/PI3K, p-AKT/AKT, p-mTOR/ mTOR ratios. Conclusion QFG can exert its effect on both tumor cells and HLECs, exhibiting ani- lymphangiogenesis in colorectal cancer via the VEGF-C/VEGFR-3 dependent PI3K/AKT pathway pathway.

BACKGROUND Qingjie Fuzheng granules (QFGs) are part of a traditional Chinese medicine formula, which has been widely used and found to be clinically effective with few side effects in various cancer treatments, including colorectal cancer (CRC). However, the precise mechanisms and molecular signaling pathways involved in the activity of QFGs’ anticancer effect have not been reported in the literature. In this study, we hypothesized that QFGs can inhibit the growth of colorectal cancer cells, and that its mechanism is closely related to one or more intracellular signal transduction pathways. AIM To better evaluate the mechanism underlying the anti-cancer effect of QFGs on the CRC cell lines HCT-116 and HCT-8. METHOD First, we measured cell viability and cytotoxicity by performing MTT and lactate dehydrogenase (LDH) assays. We evaluated the role of QFGs in cell proliferation and apoptosis by assessing colony formation and analyzing Hoechst 33258 staining. Second, cell cycle and apoptosis rates were measured by fluorescence activated cell sorting, and the expression levels of survivin, cyclin D1, CDK4, p21, Bax, Bcl-2, Fas, FasL, and cleaved-caspase-3/-8/-9 were measured by performing western blots and caspase activity assays. Furthermore, inhibitors of caspase-3/-8/-9 were used to elucidate the specific apoptosis pathway induced by QFGs in cancer cells. Finally, activation of the PI3K/AKT and ERK signaling pathways was examined using the western blot assay to investigate the possible mechanism. RESULTS MTT and LDH assays revealed that after 0.5-2.0 mg/mL of QFGs treatment, cell viability was reduced by (6.90% ± 1.03%)–(59.70% ± 1.51%) (HCT-116; P < 0.05) and (5.56% ± 4.52%)–(49.44% ± 2.47%) (HCT-8; P < 0.05), and cytotoxicity was increased from 0.52 ± 0.023 to 0.77 ± 0.002 (HCT-116; P < 0.01) and from 0.56 ± 0.054 to 0.81 ± 0.044 (HCT-8; P < 0.01) compared with the non-QFGs treatment groups. Additionally, colony formation and Hoechst 33258 staining assays showed that QFGs inhibited proliferation and induced apoptosis in CRC cells. QFGs also increased the expression levels of Bax, Fas and FasL, decreased the level of Bcl-2, and stimulated the activation of caspase-3/-8/-9, which were revealed by western blot and caspase activity assays. In contrast, when adding the three caspase inhibitors, the suppression effect of QFGs on cell viability and apoptosis were markedly inhibited. Moreover, QFGs suppressed the phosphorylation levels of PI3K, AKT and ERK. CONCLUSION These results demonstrated that QFGs can inhibit CRC cell proliferation and induce apoptosis by suppressing the PI3K/AKT and ERK signaling pathways.

Cell division cycle associated 5 (CDCA5) is implicated in the development and progression of a variety of human cancers. Functional significance of CDCA5 in colorectal cancer (CRC), however, has not been investigated. Using a combination of on-line data mining, biochemistry, and molecular biology, we examined the potential oncogenic activity of CDCA5 and the underlying mechanisms. Experiments with human tissue sample showed increased CDCA5 expression in CRC vs. in noncancerous adjacent tissue, and association of CDCA5 upregulation in CRC tissues with shorter patient survival. Also, representative CRC cell-lines had higher CDCA5 expression vs. fetal colonic mucosal cells. CDCA5 knockdown using lentivirus-mediated shRNA inhibited the proliferation and induced apoptosis in cultured HCT116 and HT-29 cells, and suppressed the growth of xenograft in nude mice. CDCA5 knockdown decreased the expression of CDK1 and CyclinB1, increased caspase-3 activity, cleaved PARP and the Bax/Bcl-2 ratio. CDCA5 knockdown also significantly decreased phosphorylation of ERK1/2 and expression of c-jun. Taken together, these findings suggest a significant role in CRC progression of CRC, likely by activating the ERK signaling pathway.

Introduction Tumor metastasis is the most important biological property of malignant tumors, and it can lead to poor prognosis. Studies have shown that transforming growth factor-β (TGF-β)/Smad signaling pathway acts an extremely important part in the process of tumor metastasis. Hedyotis diffusa Willd (HDW) is an effective anti-cancer medicine, which is accomplished in treating various types of cancer, including colorectal cancer (CRC). Nevertheless, the mechanism by which HDW inhibits metastasis of CRC is unclear. Therefore, to further clarify the molecular mechanism of its anti-CRC, we studied the effects of ethanol extract of HDW (EEHDW) on CRC cell metastasis through inhibition of TGF-β-induced EMT. Methods TGF-β stimulated CRC cell line HCT-8 cells were used, together with MTT and Transwell assays in order to assess the effect of EEHDW on the proliferation, migration and invasion of HCT-8 cells. Furthermore, the expression of markers of EMT (E-cadherin, N-cadherin, Vimentin) and TGF-β signaling pathway associated protein were detected by western blot. Results TGF-β could promote cell proliferation, moreover, TGF-β could increase the migration and invasion ability of HCT-8 cells markedly, and TGF-β induced EMT by up-regulating the expression of N-cadherin and Vimentin, down-regulating the expression of E-cadherin. The expression of related proteins (TGF-β, p-Smad2/3, Smad4) in the TGF-β/Smad pathway was increased by TGF-β. After treating with EEHDW, we observed that cell viability was reduced by EEHDW markly, inhibited the proliferation of HCT-8 cells, moreover, it could attenuate the transfer capacity of HCT-8 cells in a dose-dependent manner. Furthermore, EEHDW decreased the expression of N-cadherin, Vimentin and the proteins (TGF-β, p-Smad2/3, Smad4) in TGF-β signaling pathway, but increased the expression of E-cadherin. Conclusion EEHDW reduces migration and invasion through inhibition of TGF-β-induced EMT in colorectal cancer cells.

Summary: Background: Our previous studies reported that Qianliening capsule (QC) has a significant therapeutic effect on BPH. Therefore, we investigated the effect QC on apoptosis of human prostatic hyperplasia epithelial-1 cells (BPH-1). Methods: The BPH-1 cells were treated with various concentrations of QC in vitro. Morphology of BPH-1 cell was observed, and the cell viability was determined by the 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay. The levels of Cytochrome C, caspase-9 and caspase-3 were detected using the flow cytometry and colorimetric assay respectively. The Bax mRNA and the miRNA-221, -222, -15a, -16, -181a was determine by Real-time PCR analysis. Results: The apoptosis of BPH-1 cells treated with QC increased than that of untreated cells, as evidenced by loss of plasma membrane asymmetry, the nuclear condensation and fragmentation, collapse of mitochondrial membrane potential in a dose depended manner. The levels of Cytochrome C and caspase-9, caspase-3 in the cells treated with QC increased using the flow cytometry and colorimetric assay respectively. The mRNA and protein expression of Bax and the expression of miRNA-181a in the cells treated with QC increased in a dose dependent manner. Conclusion: QC could induce BPH-1 cells apoptosis by regulating miRNA-181a mediated mitochondrial dependent apoptosis pathway, which may be one of the important mechanisms that QC treated benign prostatic hyperplasia. Keywords: qianliening capsule, benign prostatic hyperplasia, apoptosis, miRNA181a

Qingjie Fuzheng granule (QFG) is a traditional Chinese medicinal formula used extensively as an alternative medicine for cancer treatment, including colorectal cancer (CRC). But its pathological mechanism in CRC is unclear. To study antitumor treatment effects and mechanisms of QFG, we established a CRC HCT-116 xenograft mouse model and assessed QFG on EMT and autophagy progression in vivo. The mice were randomly divided into 2 groups (n = 10 each group) and treated with intragastric administration of 1 g/kg of QFG or saline 6 days a week for 28 days (4 weeks). Body weight was measured every other day with electronic balance. At the end of the treatment, the tumor weight was measured. Immunohistochemical (IHC) and western blot (WB) assay were used to detect the expression level of E-cadherin, N-cadherin, vimentin, and TWIST1 to evaluate the effect of QFG on tumor cell EMT progression. IHC and WB assay were also used to detect the expression level of beclin-1, LC3-II, and p62 to evaluate the effect of QFG on tumor cell autophagy progression. Furthermore, the expression level of relative proteins in mTOR pathway was detected by WB assay to investigate the mechanism of QFG effect on CRC. We discovered that QFG inhibited the rise of tumor weight while it had no effect on mice body weight, which proved that QFG could inhibit CRC growth progression without significant side effects in vivo. In addition, QFG treatment inhibited EMT and induced autophagy progression in CRC tumor cells, including that QFG upregulated the expression of E-cadherin, beclin-1, and LC3-II, but downregulated the expression of N-cadherin, vimentin, TWIST1, and p62. And, QFG decreased the ratio of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR, but increased the ratio of p-AMPK/AMPK. All findings from this research proved that QFG can induce autophagy and inhibit EMT progression in CRC via regulating the mTOR signaling pathway.

Qingjie Fuzheng granule (QFG) promotes cancer cell apoptosis and ameliorates intestinal mucosal damage caused by 5-fluorouracil. However, the antitumor role of QFG in colorectal cancer (CRC) progression remains unclear. In this study, the growth of HCT-8 and HCT116 cells incubated with various concentrations of QFG for 24 and 48 h was evaluated using MTT assays; their abilities of migration and invasion were investigated through wound healing and Transwell assays. The expression of lncRNA ANRIL, let-7a, and the TGF-β1/Smad signaling pathway components was assessed using real-time PCR and western blotting. The results elicited that QFG significantly suppressed the growth of HCT-8 and HCT116 cells; the half-maximal inhibitory concentrations (IC50) of QFG for HCT-8 and HCT116 cells for 48 h were 1.849 and 1.608 mg/mL, respectively. The abilities of wound healing, migration, and invasion of HCT-8 and HCT116 cells were dose-dependently decreased by QFG treatment for 24 h, respectively. QFG decreased the expression of lncRNA ANRIL, TGF-β1, phosphorylated (p)-Smad2/3, Smad4, and N-cadherin and upregulated the expression of let-7a in HCT-8 and HCT116 cells. Collectively, our data demonstrated that QFG inhibited the metastasis of CRC cells by regulating the lncRNA ANRIL/let-7a/TGF-β1/Smad axis, indicating that they might serve as an adjunctive medicine for CRC treatment.

BackgroundAnlotinib is a multi-tyrosine kinase inhibitor that has been reported to have activity against colorectal cancer. However, the functional mechanisms whereby anlotinib mediates against deadly drug-resistant colorectal cancer (CRC) has not been fully described-specifically, the potential mechanisms that inhibit proliferation and induce apoptosis remain largely unknown.MethodsMTT assays were used to detect cell viability and calculate the resistance index. Colony formation was used to evaluate the proliferation of resistant cells. DAPI staining was used to detect cell apoptosis morphologically. Annexin V-FITC with PI staining was used to detect early and late-stage apoptosis of cells. Cell cycle distribution was determined by Flow cytometry. Transwell assays were performed to examine the ability of migration and invasion. Cyclin D1 Survivin, CDK4, Bcl-2, Bax and changes of PI3K/AKT pathway were detected by Western blotting. Compared as a single agent or combined with anlotinib or LY294002, PI3K inhibitor (LY294002) was used to verify whether it inhibited drug-resistant CRC cells by lowering PI3K/AKT.ResultsHCT-8/5-FU cells showed multiple drug resistance. Drug resistance index of 5-FU, ADM and DDP were 390.27, 2.55 and 4.57, respectively. Anlotinib was shown to inhibit cell viability on HCT-8/5-FU and HCT-8 cells for 24 h and 48 h in a dosage- and time-dependent pattern. Compared with 48 h, intervened with anlotinib (0 μM, 10 μM, 20 μM and 40 μM) for 24 h, the HCT-8/5-FU cells were sensitive to anlotinib, and their sensitivity was greater than that of the parent cell line (HCT-8) at 24 h. Further, anlotinib inhibited the number of cloned cells significantly and had a significant inhibitory effect on cell cycle, mainly by blocking G1 transferring to S phase. Moreover, anlotinib could down-regulate the expression of survivin, cyclin D1, CDK4, caspase-3, Bcl-2, MMP-2, vimentin, MMP-9, and N-cadherin, while up-regulating cleaved-caspase-3, Bax and E-cadherin. Anlotinib inhibited the activity of the PI3K/AKT pathway and induced apoptosis in HCT-8/5-FU cells. Using LY294002, a specific PI3K inhibitor, our experiment found anlotinib can inhibit drug-resistant CRC cells by reducing PI3K and p-AKT activity-induce apoptosis.ConclusionsAnlotinib inhibited the proliferation, metastasis and induced apoptosis of HCT-8 / 5-FU cells; and the mechanism could be that anlotinib overcomes multiple drug resistant of the colorectal cancer cells via inactivating PI3K/AKT pathway.

The use of 5-fluorouracil (5-FU) has been proven benefits, but it also has adverse events in colorectal cancer (CRC) chemotherapy. In this study, we explored the mechanism of 5-FU resistance by bioinformatics analysis of the NCBI public dataset series GSE81005. Fifteen hub genes were screened out of 582 different expressed genes. Modules of the hub genes in protein-protein interaction networks gathered to TOP2α showed a decrease in HCT-8 cells but an increase in 5-FU-resistant HCT-8/5-FU cells with 5-FU exposure. Downregulation of TOP2α with siRNA or miR-494 transfection resulted in an increase of cytotoxicity and decrease of cell colonies to 5-FU for HCT-8/5-FU cells. Moreover, we found that an ethanol extract of Spica Prunellae (EESP), which is a traditional Chinese medicine with clinically beneficial effects in various cancers, was able to enhance the sensitivity of 5-FU in HCT-8/5-FU cells and partly reverse the 5-FU resistance effect. It significantly helped suppress cell growth and induced cell apoptosis in HCT-8/5-FU cells with the expression of TOP2α being significantly suppressed, which increased by 5-FU. Consistently, miR-494, which reportedly regulates TOP2α, exhibited reverse trends in EESP/5-FU combination treatment. These results suggested that Spica Prunellae may be beneficial in the treatment of 5-FU-resistant CRC patients.

To investigate the protective effects of Shexiang Tongxin Dropping Pill (麝香通心滴丸, STP) on Na2S2O4-induced hypoxia-reoxygenation injury in cardiomyoblast H9c2 cells. The cell viability and levels of mRNA and protein expression in H9c2 cells were determined following Na2S2O4-induced hypoxia using Hoechst staining, annexin V/propidium iodide (PI) flow cytometry, real-time polymerase chain reaction and Western blot analysis. STP pretreatment significantly increased the viability and inhibited aberrant morphological changes in H9c2 cardiomyoblast cells induced by Na2S2O4 treatment (P

Ursolic acid (UA) has shown great potential in cancer therapy but their efficacy is seriously compromised by poor water-solubility and limited cellular uptake. In this paper, cationic nanomicelles self-assembled from Pluronic F127 with the cationic polymer of C18-polyethylenimine (C18-PEI) as a functional component are fabricated as delivery vehicles of Chinese herbal medicine active components of ursolic acid (UA) for colorectal cancer treatment. The inhibition effects of this drug loaded cationic nanomicelles (named as FUP) on cell viability and cell colony formation were more significant than the free UA, due to their cationic surface which can increase UA uptake by colorectal cancer cells. Cell cycle analysis showed that this inhibition effect was mediated by a cell cycle arrest at the G1 checkpoint, and the cell death induced by these nanomicelles occurred via apoptosis, which was detected by annexin V antibody and propidium iodide staining. Further western blot analysis demonstrated the apoptosis mechanism was associated with the regulation of Fas/FasL and activation of caspase-8 and caspase-3. Therefore, our cationic nanomicelles can potentially be used to enhance the therapeutic effect of UA for colorectal cancer treatment.

Ba-Bao-Dan (BBD) is a well-known Traditional Chinese medicine (TCM) prescription in China. It was first formulated in approximately 1555 AD. As one of the National Protected TCM, it is widely used to treat jaundice, viral hepatitis, cholecystitis, acute urinary tract infection, cancer, and other diseases. It is a healthcare medicine that is used to prevent many diseases in China. In other Asian countries and in European and American countries, BBD is used as a drug to protect the liver. However, a systematic quality study on BBD chemical markers has not been carried out. This study aimed to establish an ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS) method for the quantitative determination of 43 compounds in BBD. Furthermore, the method was used to further find chemical markers for quality control through the combination with chemometrics. The modified chromatographic conditions were achieved on Waters Cortecs C18 column (2.1 × 100 mm, 1.6 μm) with a gradient elution consisting of 0.1 % formic acid in water and acetonitrile with methanol (1:1, V/V). All analytes were determined in the multiple reaction monitoring mode. The method was validated for linearity, detection limits, precision, repeatability, stability and accuracy. The method was used to analyze the 43 compounds in 11 batches of BBD samples. Hierarchical cluster analysis and principal component analysis were applied to evaluate intrinsic quality of BBD and to identify the potential chemical markers for quality control. In conclusion, the method rapidly and sensitively determined the 43 compounds, among which 10 compounds, namely, N-Gin R1, Gin Re, Gin Rg1, Gin Rb1, GCA, Gin Rd, CA, TCA, CDCA, and DCA, were considered as the potential chemical markers for BBD quality control.

OBJECTIVE To investigate the effect of the ethanol extract of Scutellaria barbata D. Don (EESB) on colorectal cancer (CRC) growth and Wnt/β-catenin signaling pathway in vivo and in vitro. METHODS In vivo experiment, CRC xenograft mouse model was constructed with injection of HT-29 cells. Following xenograft implantation, twenty mice were randomly divided into EESB-treated group (n=10) and control group (n=10) by a random number table, and were given with intra-gastric administration of 2 g/kg EESB or saline, 5 days a week for 16 days, respectively. At the end of experiment, tumors were removed and weighed by electronic scales. The proliferation biomarker Ki-67 of tumor was evaluated by immunohistochemistry (IHC) assay. In vitro study, HT-29 cells were treated with 0, 0.5, 1.5, 2.5 mg/mL EESB for 24 h. At the end of the treatment, the viability and survival of HT-29 cells were determined by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and colony formation assay, respectively. The mRNA expression of c-Myc, Survivin and adenomatous polyposis coli (APC) was examined by reverse transcription-polymerase chain reaction (RT-PCR) both in tumor tissues of CRC xenograft mice and HT-29 cells. Protein expression of c-Myc, Survivin, APC, and β-catenin as well as β-catenin phosphorylation level were evaluated by IHC assay or Western blotting. RESULTS EESB significantly reduced tumor weight in CRC xenografts mice, compared with the control group (P

Chemotherapeutic agents, including 5-fluorouracil (5-FU), frequently cause intestinal mucositis as a side effect, leading to life quality reduction in colorectal cancer (CRC) patients and interruption of CRC treatment. Traditional Chinese medicines (TCMs) have recently received attention due to their relatively few adverse effects. Pien Tze Huang (PZH), a well-known TCM formulation first documented to have been prescribed >450 years ago, has been demonstrated to be clinically effective in treating various types of cancer including CRC. Although previous studies by our group reported that PZH possesses a variety of anti-cancer activities via multiple mechanisms, it has remained elusive whether it is able to reduce intestinal mucositis induced by 5-FU. The present study evaluated the effect of PZH on 5-FU-induced intestinal mucositis in CT-26 tumor-bearing xenograft mice and investigated the possible molecular mechanism. The results indicated that administration of PZH effectively alleviated the severity of 5-FU-induced diarrhea and morphological intestinal damages, but had no significant effect on body weight loss. In addition, a terminal deoxynucleotidyl transferase dUTP nick end labeling assay revealed that PZH treatment significantly inhibited cell apoptosis in the intestinal crypt. Furthermore, immunohistochemical staining showed that PZH treatment reduced the protein expression of pro-apoptotic B-cell lymphoma 2 (Bcl-2)-associated X protein but enhanced that of anti-apoptotic Bcl-2 in the intestinal crypt. Taken together, the results of the present study suggested that PZH effectively attenuates 5-FU-induced intestinal mucositis, which is in part associated with its inhibitory effect on cell apoptosis in the intestinal crypt.

5-Fluorouracil (5-FU) resistance or multidrug resistance (MDR) has become a major obstacle in clinical treatment of cancers including colorectal cancer (CRC). Aberrant activation of phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) pathway may lead to unlimited growth and chemoresistance in CRC cells, which thus could be a promising therapeutic target. As a long-term used traditional Chinese folk-medicine, Scutellaria barbata D. Don (SB) processes specific anticancer activity, but its activity against cancer chemoresistance is less known. Therefore, using a 5-FU-resistant CRC cell line HCT-8/5-FU, in this study we evaluated the therapeutic efficacy of the ethanol extracts of SB (EESB) against 5-FU resistance and explored the possible molecular mechanisms. We found that EESB significantly suppressed proliferation and promoted apoptosis in HCT-8/5-FU cells. Additionally, EESB displayed remarkable effect enhancing the retention of the ATP-binding cassette (ABC) transporter substrate, rhodamine‑123 (Rh‑123) in HCT-8/5-FU cells. Furthermore, EESB obviously downregulated the expression of cyclin D1, Bcl-2 and ABCG2, while upregulated p21 and Bax expression. Moreover, EESB showed a prominent suppressive effect on the activation of PI3K/AKT pathway. The findings suggested that Scutellaria barbata D. Don was able to inhibit chemoresistance in colorectal cancer by suppression of the PI3K/AKT pathway.