Evaluation of Antiproliferative Activity, Safety and Biodistribution of Oxaliplatin and 5-Fluorouracil Loaded Lactoferrin Nanoparticles for the Management of Colon Adenocarcinoma: an In Vitro and an In Vivo Study.

Inflammation is known to promote colorectal cancer (CRC) tumorigenesis, but the underlying molecular mechanisms are still being uncovered. Proinflammatory cytokine interleukin-6 (IL-6) stimulates growth and survival signaling in CRC cells. Inflammatory signals regulate also the production and activity of proteases and their inhibitors. Serine protease inhibitor Kazal type1 (SPINK1, aka tumor-associated trypsin inhibitor, TATI) is expressed in several tissues and over-expression of SPINK1 predicts an unfavorable outcome in many cancers, including colon cancer. The SPINK1 gene contains an IL-6 responsive element and in addition to being a protease inhibitor, it also acts as an acute phase reactant and a growth factor. The aim of this study was to assess the connection between IL-6 and SPINK1 and the effect of this signaling to growth and invasion of CRC cells. The following expression analyses and functional assays were used: quantitative PCR, immunohistochemistry, immunofluorometric assays, Western blotting, cell proliferation and migration assays. The results of this study show that CRC cell lines Colo205 and HT-29 express SPINK1 and secrete it into the culture medium. In Colo205 and HT-29 cells IL-6 dose-dependently increased the mRNA expression and protein levels of SPINK1. The baseline secretion was lower in the Colo205 cells, and significantly more IL-6 was required for the dose-dependent induction of SPINK1 expression in these cells as compared to HT-29 cells. Interestingly, in Colo205 the baseline levels of secreted tumor-associated trypsinogen-1 and -2 (TAT-1 and -2), the key targets of SPINK1 protease inhibitory action, were much higher than in HT-29 cells. In these cells IL-6 led to concomitant increase in the secretion of TAT-1 and -2, whereas in HT-29 cells the TAT-1 and -2 levels remained constantly low. IL-6 at concentrations giving the maximum SPINK1 induction did not increase the proliferation of Colo205 or HT-29 cells; however the motility of these cells was increased in response to this proinflammatory cytokine. Mechanistically, addition of IL-6 led to activation of the canonical signal transducer and activator of transcription 3 (Stat3) pathway, as indicated by Stat3 phosphorylation in both Colo205 and HT-29 cells. Taken together, our results indicate a connection between inflammatory response and increased SPINK1 levels. Elevated serum SPINK1 has been shown to be an independent prognostic factor in colon cancer. As SPINK1 acts as a growth factor in some cancers, it has also been suggested as a therapeutic target. Therefore assessment of SPINK1 expression and function has several potentially important clinical applications in colorectal and other cancers. Citation Format: Kati A. Räsänen, Laura Hautala, Ulf-Håkan Stenman, Hannu Koistinen. Interleukin-6 induces SPINK1 in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 439. doi:10.1158/1538-7445.AM2015-439

Purpose : This study aimed to evaluate the possible cytotoxic activity of the essential oil of the Eugenia uniflora leaves on normal and tumoral human cells by colorimetric cell viability assay, based on the use of tetrazolium salt (XTT). Methods : To achieve the cytotoxicity it was used normal human lung fibroblast cell line GMO7492A and the evaluation of antiproliferative activity was performed on three tumor cell lines, as follows: human glioblastoma (MO59J), human cervical adenocarcinoma (HeLa) and human breast adenocarcinoma (MCF-7). For the determination of cytotoxic concentration to the normal line, 12 concentrations were evaluated being from 2.44 to 5000 µg/mL. In the evaluation of antiproliferative activity were tested 8 different concentrations of the extract (3.91 to 500 µg/mL). Results : The results in the normal line GM07492A showed that concentrations higher or equal to 39.1 µg/mL are significantly different of the negative control. In the evaluation of antiproliferative activity on tumor cell lines MO59J, HeLa and MCF-7 was observed that the concentration of 125 µg/mL showed a cytotoxic effect on these lines being significantly different from the negative control. The results from the evaluation of the antiproliferative activity in different tumor cell lines of E. uniflora oil was not selective for the tested cell types. Conclusion : E. uniflora oil did not show cytotoxic activity in concentrations lower than 39.1 µg/mL, however, the values found to IC 50 for tumor cells were superior, concluding that the oil has no selectivity for tumor cells tested.

In vitro antiproliferative activity of 2,3-dihydroxy-9,10-anthraquinone induced apoptosis against COLO320 cells through cytochrome c release caspase mediated pathway with PI3K/AKT and COX-2 inhibition

Thyroid hormone has been shown to induce proliferation of a variety of human cancer cell lines via a cell surface binding site on integrin αvβ3. Clinical studies indicate that thyroid hormone may support the growth of brain tumor, breast cancer, head-and-neck cancer, renal cell carcinoma and several other cancers. The effect of microenvironmental thyroid hormone on the growth of colorectal cancer has not been investigated fully. Numerous evidences suggest that the Wnt/β-catenin pathway, which is involved in both normal and oncogenic developments of the gut, is associated with thyroid hormone and its downstream signaling. The study is therefore aimed to look at the role of thyroid hormone, thyroxine (T4) on colon cancer progression through β-catenin activation. Materials and Methods: The effect of T4 in two APC mutant colon cancer cell lines HT-29 and Colo205 cells and one APC wild type colon cancer HCT116 cells were studied. MTT assays were performed to evaluate cell proliferation under different concentrations of T4. Relative mRNA and protein expressions of relevant genes were examined using qPCR and Western blotting, respectively. Results: T4 (10−8 to 10−6 M) induced proliferation in colorectal cancer HT-29 cells, HCT116 cells and Colo205 cells in a concentration-dependent manner. In HT29 and Colo205 cancer cell lines, the expressions of β-catenin and HMGA2 were not significantly altered by the application of T4. Alternatively, T4 increased accumulation of nuclear β-catenin and HMGA2 in HT-29 cells. Nuclear β-catenin and message were elevated by T4 in Colo205 cells. On the other hand, in addition to the cell proliferation and nuclear accumulation of β-catenin and HMGA2, T4 (10−8-10−6 M) induced the expression of β-catenin and HMGA2 in HCT116 cells. The expression of genes involved in proliferation was enhanced by thyroxine treatment. Conclusions: T4 promoted proliferation in colon cancer HT-29, Colo205 and HCT116 cells. The effect of T4 in colon cancer involved nuclear distribution of β-catenin and its activation. However, the different phenotypes of APC affected the accumulation of nuclear β-catenin and HMGA2 induced by T4. Further study concerning detailed mechanisms is required to elucidate the role of thyroid hormone in colon cancer progression. By understanding the mechanisms behind the pathogenic effects of thyroid hormone, we will propose and develop new treatment approaches to colorectal cancers. Citation Format: Hung-Yun Lin. Thyroid hormone induced Beta-catenin-dependent proliferation in colorectal cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C96.

Docetaxel, a chemotherapeutic agent currently used for improving survival of prostate cancer patients, suffers from low therapeutic index. The objective of this study was to prepare a new docetaxel derivative conjugated to deslorelin, a luteinizing hormone-releasing hormone (LHRH) superagonist, and to determine whether it enhances docetaxel potency in vitro and in vivo. Because docetaxel is not amenable for conjugation with peptides, we introduced a -COOH group in docetaxel, forming docetaxel-hemiglutarate, and subsequently conjugated this to serine in deslorelin, forming deslorelin-docetaxel. Fourier-transform IR, (1)H-nuclear magnetic resonance, and liquid chromatography-mass spectrometry analyses confirmed deslorelin-docetaxel formation. Antiproliferative efficacy in LNCaP and PC-3 cell lines over 24, 48, and 72 hours exhibited the order deslorelin-docetaxel > docetaxel, whereas deslorelin alone had no effect, with deslorelin-docetaxel potency being 15-fold greater than docetaxel at 72 h. Further, cells pretreated with antisense oligonucleotide against LHRH receptor exhibited decreased deslorelin-docetaxel efficacy, without any change in docetaxel efficacy. Thus, deslorelin-docetaxel efficacy is likely mediated via LHRH receptor. Cell cycle analysis showed that docetaxel treatment led to arrest in G(2)-M phase, whereas deslorelin-docetaxel treatment allowed greater progression to apoptosis in both cell lines, with deslorelin-docetaxel exerting 5-fold greater apoptosis compared with docetaxel in prostate cancer cell lines. Antitumor efficacy studies in PC-3 prostate xenograft-bearing mice indicated the efficacy order deslorelin-docetaxel > docetaxel >> deslorelin > PBS, with deslorelin-docetaxel exerting approximately 5.5-fold greater tumor growth inhibition than docetaxel alone. Thus, deslorelin-docetaxel prepared in this study retains pharmacologic effects of both docetaxel and deslorelin while enhancing the antiproliferative, apoptotic, and antitumor efficacy of docetaxel by several folds in prostate cancer therapy.

Among 13 different cell lines, gossypol (GOS) showed the most potent cytotoxic effect against human colorectal carcinoma cells including HT29, COLO205, COLO320HSR and COLO320DM cells according to an MTT assay. The cytotoxic effect of GOS was mediated by its induction of apoptosis as characterized by the occurrence of DNA ladders, apoptotic bodies and chromosome condensation in both COLO205 and HT29 cells. Activation of caspase 3, 6, 8 and 9, but not caspase 1, accompanied by the appearance of cleaved fragments of PARP (85 kDa), and caspase 3 (p17/p15), was identified in GOS-treated cells. Decreases in Bcl-xL and phosphorylated Bad proteins were found in GOS-treated cells. GOS induction of ROS production was detected by in vitro plasmid digestion, and an increase in the intracellular peroxide level was observed in GOS-treated COLO205 cells by the DCHF-DA assay. Antioxidants including N-acetyl-L-cysteine (NAC), catalase (CAT), tempol (TEM) and melatonin (MEL), but not allopurinol (ALL), pyrrolidine dithiocarbamate (PDTC) or diphenylene iodonium (DPI), significantly inhibited GOS-induced Reactive oxygen species (ROS) production through blocking the occurrence of apoptosis. GOS induced mitochondrial dysfunction characterized by a loss of the mitochondria membrane potential via DiOC6 staining, and the release of cytochrome c (Cyt c) and apoptosis-inducing factor (AIF) from mitochondria to the cytoplasm was observed. Removing mitochondria by ethidium bromide (EtBr) treatment significantly reduced the apoptotic effect of GOS in COLO205 cells. Furthermore, an intraperitoneal injection of GOS or gossypol acetic acid (GAA) significantly reduced the growth of colorectal carcinoma induced by a subcutaneous injection of COLO205 cells in nude mice. Results of the present study provide the first evidences demonstrating the in vitro and in vivo antitumor effects of GOS via an ROS-dependent mitochondrial apoptosis in colorectal carcinoma.

Context Ambrosia artemisiifolia L. (Asteraceae) contains sesquiterpene lactones as characteristic secondary metabolites. Many of these compounds exert antiproliferative and cytotoxic effects. Objective To isolate the sesquiterpene lactones from the aerial part of A. artemisiifolia and to elucidate their cytotoxic, antiproliferative and antibacterial effects. Materials and methods The compounds were identified by one-dimensional (1D) and 2D NMR, HR-MS spectroscopy from the methanol extract. Isolated compounds were investigated for their cytotoxic and antiproliferative effects on human colonic adenocarcinoma cell lines and human embryonal lung fibroblast cell line using MTT assay. The selectivity of the sesquiterpenes was calculated towards the normal cell line. To check the effect of drug interactions between compounds and doxorubicin, multidrug-resistant Colo 320 cells were used. Results A new seco-psilostachyinolide derivative, 1,10-dihydro-1′-noraltamisin, and seven known compounds were isolated from the methanol extract. Acetoxydihydrodamsin had the most potent cytotoxic effect on sensitive (Colo205) cell line (IC50 = 7.64 µM), also the strongest antiproliferative effect on Colo205 (IC50 = 5.14 µM) and Colo320 (IC50 = 3.67 µM) cell lines. 1′-Noraltamisin (IC50 = 8.78 µM) and psilostachyin (IC50 = 5.29 µM) showed significant antiproliferative effects on the multidrug-resistant Colo320 cell line and had moderate selectivity against human embryonal lung fibroblast cell line. Psilostachyin C exhibited cytotoxic effects on Colo205 cells (IC50 = 26.60 µM). None of the isolated compounds inhibited ABCB1 efflux pump (EP; P-glycoprotein) or the bacterial EPs. Discussion and conclusions Acetoxydihydrodamsin, 1′-noraltamisin, and psilostachyin showed the most remarkable cytotoxic and antiproliferative activity on tumour cell lines and exerted selectivity towards MRC-5 cell line.

Colon adenocarcinoma (COAD) is one ofthemost prevalent forms ofcancer in theworld. Still, themolecular mechanism ofCOAD development remains unknown, making it especially important to investigate themolecular mechanism ofCOAD development and identify new therapeutic targets. Areal-time fluorescence quantification polymerase chain reaction (RT-qPCR) was used to determine thelevel ofmiR-21-5p expressionin COAD tissues and cell lines. Both miR-21-5p silencing and overexpression were performed in LOVO and T84 cell lines. Cell viability, apoptosis rate, migration, and invasion ability were determined using MTT, flow cytometry, and theTranswell assay, respectively. Western blot was applied to detect thelevels ofprotein expression associated with theepithelial-mesenchymal transition (EMT). Using adual luciferase reporter gene, thetargeting connection among miR-21-5p and Tensin 1 was validated. Tensin 1 expression was silenced to investigate its effect on miR-21-5p inhibitor activity in COAD cells. Subcutaneous tumor-forming animal experiments in nude mice were used to investigate theeffect ofmiR-21-5p on COAD tumor growth in vivo. Ki-67 expression was identified through immunohistochemistry. MiR-21-5p was found in high concentrations in COAD tissues and cells. Overexpression ofmiR-21-5p increased COAD cell line viability and EMT, facilitated cell migration and invasion, and inhibited apoptosis. Tensin 1 was regulated negatively by miR-21-5p. Tensin 1 silencing reversed theeffect ofmiR-21-5p silencing on COAD cells. Subcutaneous tumor formation experiments in nude mice revealed that inhibiting miR-21-5p expression slowed the growth rate of tumor volume. According to immunohistochemical results, thepercentage ofKi-67-positive cells was significantly lower in theanti-miR-21-5p group. MiR-21-5p levels were upregulated in COAD cells, and reducing miR-21-5p expression inhibited COAD cell viability, migration, invasion, and EMT in vitro. Tensin 1 negatively regulated miR-21-5p, which regulated COAD cell and EMT progression.

Development of drug resistance in cancer is one of the main challenges in chemotherapy, and many mechanisms are still unknown. In this study, we show that tumor necrosis factor alpha (TNFalpha) increases postdrug survival from 5-fluoro-2'-deoxyuridine (FdUrd) in two human colon tumor cell lines. This resulted in the development of drug-resistant cells in a TNFalpha-dependent manner. Interestingly, although the drug-resistant cells were selected using FdUrd, they are also resistant to a number of other antimetabolites in the DNA synthesis pathway in a TNFalpha-dependent manner. Only in the drug-resistant cells (p35-colo201) TNFalpha treatment resulted in G(0)-G(1) arrest but not in the parental colo201 and other cell types. Blocking TNFalpha-induced cell cycle arrest sensitized drug-resistant cells to FdUrd. TNFalpha-induced cell cycle arrest required IKK. IKK inhibition by a small molecule inhibitor or by the knockdown of IKKalpha, IKKbeta, or RelA/p65 using siRNA, but not the inhibition of JNK, MEK, p38, or caspase-8 pathways, blocked TNFalpha-induced G(0)-G(1) arrest and restored sensitivity to FdUrd of drug-resistant cells. TNFalpha reduced the transcripts and protein levels of phosphorylated retinoblastoma protein (Rb), Rb, E2F1, and Cdk4 only in drug-resistant p35-colo201 cells. This effect of TNFalpha was reversed by IKK inhibitor, suggesting that TNFalpha-induced cell cycle arrest is probably due to the reduction of Rb, E2F1, and Cdk4. Taken together, this study shows that, in vitro, TNFalpha-induced cell cycle arrest through IKK can provide a mechanism for the development of drug resistance to anti-cancer drugs, purine and pyrimidine analogues.

Apoptosis, or programmed cell death, plays an important role in many physiological and diseased conditions. Induction of apoptosis in cancer cells by anti-cancer drugs and biosynthetic inhibitors of cells surface glycolipids in the human colon carcinoma cells (Colo-205) are of interest in recent years. In our present studies, we have employed different stereoisomers of PPMP and PDMP (inhibit GlcT-glycosyltransferase (GlcT-GLT)) to initiate apoptosis in Colo-205 cells grown in culture in the presence of (3)H-TdR and (3)H/or (14)C-L-Serine. Our analysis showed that the above reagents (between 1 to 20 microM) initiated apoptosis with induction of Caspase-3 activities and phenotypic morphological changes in a dose-dependent manner. We have observed an increase of radioactive ceramide formation in the presence of a low concentration (1-4 microM) of these reagents in these cell lines. However, high concentrations (4-20 microM) inhibited incorporation of radioactive serine in the higher glycolipids. Colo-205 cells were treated with L-threo-PPMP (0-20 microM) and activities of different GSL: GLTs were estimated in total Golgi-pellets. The cells contained high activity of GalT-4 (UDP-Gal: LcOse3Cer beta 1-4galactosyltransferase), whereas negligible activity of GalT-3 (UDP-Gal: GM2 beta 1-3galactosyltransferase) or GM2-synthase activity of the ganglioside pathway was detected. Previously, GLTs involved in the biosynthetic pathway of SA-Le(x) formation had been detected in these colon carcinoma (or Colo-205) cells (Basu M et al. Glycobiology 1, 527-35 (1991)). However, during progression of apoptosis in Colo-205 cells with increasing concentrations of L-PPMP, the GalT-4 activity was decreased significantly. These changes in the specific activity of GalT-4 in the total Golgi-membranes could be the resultant of decreased gene expression of the enzyme.

Objective The activity of NEK6 is enhanced in several cancer cells, including colon adenocarcinoma (COAD) cells. However, there are few reports on the microRNA (miRNA/miR) regulation of NEK6. In this study, we aimed to investigate the effects of miRNAs targeting NEK6 in COAD cells. Methods Public data and online analysis sites were used to analyze the expression levels of NEK6 and miR-323a-3p in COAD tissues as well as the relationship between NEK6 or miR-323a-3p levels and survival in patients with COAD and to predict miRNAs targeting NEK6. Real-time polymerase chain reaction and western blotting were performed to determine the levels of NEK6 and miR-323a-3p in COAD cells. The targeting of NEK6 by miR-323a-3p was verified using a dual-luciferase reporter assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, 5-ethynyl-2′-deoxyuridine assay, propidium iodide (PI) staining, annexin V-fluorescein isothiocyanate/PI staining, and transwell assay were employed to test the proliferation, apoptosis, migration ability, and invasiveness of COAD cells. Results In COAD cells, NEK6 was highly expressed, whereas miR-323a-3p was expressed at low levels and negatively regulated NEK6. Upregulating the level of miR-323a-3p impaired the proliferation, migration, and invasion of COAD cells and promoted apoptosis, whereas supplementing NEK6 alleviated the damage of the proliferation, migration, and invasion of COAD cells caused by miR-323a-3p and inhibited miR-323a-3p-induced apoptosis. These findings indicate that miR-323a-3p regulates the proliferation, migration, invasion, and apoptosis of COAD cells by targeting NEK6. Conclusion miR-323a-3p downregulates NEK6 in COAD cells; this provides a novel basis for further understanding the occurrence and development of COAD.

Chemotherapy using 5-fluorouracil (5-FU) is currently considered the most effective treatment for advanced colon adenocarcinoma (COAD). However, drug resistance remains a major obstacle in treating COAD. Non-SMC condensin I complex subunit H ( NCAPH ) is known to have a certain impact on the development of COAD, but its precise involvement in the mechanism of 5-FU resistance has not been demonstrated. Bioinformatics analysis was utilized to assay the expression of NCAPH and Forkhead box M1 ( FOXM1 ) in COAD tumor tissues, which was then verified in COAD cell lines. The resistance of COAD cells to 5-FU was measured by CCK-8 assay, stemness was tested by cell sphere formation assay, and glycolysis ability was measured by cellular energy analysis metabolism. Chromatin Immunoprecipitation and dual-luciferase reporter assays were done to confirm the specific interaction between FOXM1 and NCAPH . The expression levels of FOXM1 and NCAPH were significantly upregulated in COAD tissues and cells, and they were involved in regulating the glycolytic signaling pathway. Inhibition of the glycolytic pathway could reverse the effect of NCAPH overexpression on COAD stemness and resistance. FOXM1 was identified as a transcription factor of NCAPH , and it regulated COAD glycolysis, cell stemness, and 5-FU resistance by activating NCAPH expression. FOXM1-mediated upregulation of NCAPH expression promoted COAD cell stemness and resistance via the glycolytic pathway. This study provides a possible mechanism for the FOXM1/NCAPH axis in the glycolytic pathway, cell stemness, and resistance in COAD.

Chimeric antigen receptor-T (CAR-T) cell therapy is a cellular immunotherapy that has emerged in recent years, and increasing studies showed that therapeutic resistance to CAR-T cell therapy presents in colorectal cancer patients. Lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, is known to preserve a high concentration in the colon. Whether LPS is a contributing factor to the development of resistance in colorectal cancer cells against CAR-T cell therapy remains unclear. For in vivo experiments, colorectal cancer cells COLO205 were pretreated with LPS for 24 h and then were injected into nude mice through the tail vein, followed by CAR-T cells transplantation one day later. Later, the number of tumors in the lung tissues of the mice was observed. The in vitro experiments were performed on COLO205 cells, which were treated with LPS for 24 h. The effect of LPS on the stemness of COLO205 and SW620 cells was observed by using the colony formation assay and spheroidization experiments. The effect of LPS on the expression of stemness-related genes, including CD44, SOX2, and NANOG, was observed by qRT-PCR assay, Western blotting assay, and immunofluorescence staining. Inhibitors of TGF-β and the MYD88 inhibitor were used to study the mechanisms by which LPS induces the stemness enhancement and resistance to CAR-T cell therapy of COLO205 cells. The correlation between MYD88 and TGFB1, as well as the correlation between TGFB1 and stemness-related genes was analyzed using the TCGA database. Both the in vivo assay of nude mice and the in vitro assay showed that LPS pretreatment could induce resistance to CAR-T cell therapy of colorectal cancer cells. LPS could enhance COLO205 and SW620 cells stemness presented by upregulation of CD44, SOX2, and NANOG. The reverse interfering assay using the TGF-β inhibitor indicated that the autosecretion of TGF-β induced by LPS played a critical role in the stemness enhancement of colorectal cancer cells. The TCGA database analysis revealed a strong positive correlation between MYD88 and TGFB1. Additionally, TGFB1 has been found to upregulate the expression of genes associated with stemness. Further mechanism studies showed that the TLR4/MYD88 pathway medicates LPS-induced TGF-β expression. Our results suggested that LPS-induced resistance to CAR-T cell therapy of colorectal cancer cells through stemness enhancement. TLR4/MYD88 signal pathway-dependent TGF-β expression was involved in stemness enhancement and CAR-T cell therapy resistance. In conclusion, our findings help us to understand the underlying mechanisms of CAR-T cell therapy resistance and indicate that inhibitors of TGF-β and MYD88 are promising targeting candidates to promote a therapeutic effect of CAR-T cell therapy in colorectal cancer in the clinic.

BackgroundThe mastic gum resin has been used in traditional Kurdish medicine for treating various disorders such as topical wound and gastric ulcer. The study designed to evaluate the total polyphenol and flavonoid content, free radical scavenging activity, and anticancer effects of mastic gum resin derived from Pistacia atlantica subspecies kurdica.Materials and methodsFolin -Ciocalteau and the aluminum chloride colorimetric assays were used to determine the total phenol and flavonoid contents in the mastic gum resin respectively. Whereas, DPPH and ABTS+ assays were used to determine the antioxidant activities of mastic gum resin. Regarding anticancer activities, the MTT assay was used to study the effect of mastic gum resin on the proliferation of various cancer cells and the morphological changes were identified after Acridine Orange/Propidium Iodide staining. Flow cytometry was applied to determine the influence of mastic gum resin on the apoptosis rate by Annexin V double staining and to investigate the influence on cell cycle progression. Caspase colorimetric assay was used to estimate the hallmark enzyme of apoptosis, and finally RNA were obtained from COLO205 cells and analyzed by qRT-PCR analyses.ResultsThe MTT results showed that the mastic gum resin at concentrations from 0.01 to 100 μM induced death of cancer cells in a dose and time-dependent manner. The mastic gum resin suppressed proliferation of human cancer cells with 72 h IC50 value of 15.34 ± 0.21, 11.52 ± 0.18, 8.11 ± 0.23 and 5.2 ± 0.8 μg/mL for bile duct cancer (cholangiocarcinoma) (KMBC), pancreatic carcinoma (PANC-1), gastric adenocarcinoma (CRL-1739), and colonic adenocarcinoma (COLO205) cells, respectively. Normal human colon fibroblast (CCD-18Co) cells were not adversely affected by resin treatment. Flow cytometry showed that the mastic gum resin significantly (P<0.05) arrested COLO205 cell proliferation at the G2/M phase of cell cycle. The resin caused apoptotic morphological changes in COLO205 cells. The apoptotic effect to mastic gum resin was via the mitochondrial as shown by the up-regulation of Bax, down-regulation of Bcl-2 genes, and activation of caspase-9 and -3 activities.ConclusionIt was confirmed that the antiproliferative efficacy of the resin is positively correlated with its polyphenolic contents, suggesting a causal link related to exudate content of phenolic acid and flavonoids. The results revealed that the mastic gum resin has potential to be developed as an anticancer and antioxidant product due to its high content of polyphenol compounds.

IntroductionOdd-skipped related transcription factor 1 (OSR1) is a newly identified tumor suppressor in many tumor types. However, the role and mechanism of OSR1 in colon adenocarcinoma (COAD) remain unknown.MethodsOSR1 expression was detected in COAD tissues and cells. COAD cells with OSR1 overexpression or knockdown were analyzed by in vitro CCK-8, transwell and flow cytometry assays, and by in vivo xenograft model.ResultsOSR1 expression was downregulated in COAD and low expression level of OSR1 was positively correlated with tumor stage and lymph node metastasis. Furthermore, low OSR1 expression was significantly associated with poor overall survival (OS) and distant metastasis-free survival (DMFS). Lentivirus-mediated restoration of OSR1 expression-inhibited proliferation, invasion and migration while induced cell cycle arrest and apoptosis in COAD cells in vitro, and inhibited tumor growth in vivo. In contrast, OSR1 knockdown promoted proliferation, invasion and migration in COAD cells in vitro. Mechanistically, OSR1 exerted anticancer effects by inhibiting FAK-mediated activation of Akt and MAPK pathways.ConclusionOur findings suggest that OSR1 functions as a tumor suppressor in COAD by suppressing FAK-mediated activation of Akt and MAPK pathways.