WiDr Cells Research Articles

Anticancer effects of fucoxanthin and fucoxanthinol on colorectal cancer cell lines and colorectal cancer tissues.

Colorectal cancer is one of the most malignant neoplasms worldwide. Fucoxanthin is a carotenoid present in the chloroplasts of brown seaweeds. In the present study, the anticancer effects of fucoxanthin and its metabolite, fucoxanthinol, on 6 colorectal cancer cell lines and 20 tissue samples from surgically resected clinical colorectal cancer specimens were examined using a collagen-gel droplet embedded culture drug sensitivity test (CD-DST). The in vitro sensitivity to fucoxanthin, fucoxanthinol and the anticancer drugs is expressed as T/C (%), where T is the absorbance of cells which stained by neutral red treated with carotenoids and C is the absorbance of non-staining cells. Fucoxanthin and fucoxanthinol decreased the T/C (%) of Caco-2, WiDr, HCT116, and DLD-1 cell lines at doses of 20 µM. Fucoxanthinol also decreased the T/C (%) of SW620 cells, while the T/C (%) of Colo205 cells was not reduced by treatment with either carotenoid. Specifically, the T/C (%) of Caco-2 and WiDr cells, which were incubated in carotenoid-free medium for 6 days following treatment with 20 µM fucoxanthinol for 24 h, was markedly decreased to 1.4±0.2 and 12.0±0.3%, respectively. Furthermore, fucoxanthin and fucoxanthinol decreased the T/C (%) in colorectal cancer tissue samples. Notably, 20 µM fucoxanthinol treatment resulted in a higher proportion of colorectal cancer samples with a T/C (%) of <50% (13/20, 65%) compared with samples treated with 20 µM fucoxanthin (2/20, 10%). The median T/C (%) value of 35.1% for the 20 cancers specimens treated with 20 µM fucoxanthinol was lower than the median T/C (%) values of 86.3% and 75.8% for those treated with fluorouracil and paclitaxel, respectively. These results suggested that fucoxanthin and fucoxanthinol may be of use as chemotherapeutic agents in colorectal cancer.

The Anticancer Activity of Marine Sponge Cinachyrella sp. (Family Tetillidae)

Cinachyrella sp. marine sponge produce many kinds of secondary metabolites. The purpose of this study was to examine the cytotoxic and anti-proliferative activity of marine sponge Cinachyrella sp. The sponge was extracted with 96 % ethanol. Ethanol extract cytotoxicity assay were performed with MTT method (Microculture Tetrazolium) against to cell lines of HeLa, T47D, WiDr and Vero. The results show that of the ethanol extract were only toxic to cell lines of HeLa IC50 897.809 μg/mL but did not toxic to cell lines of T47D, WiDr and normal cell lines of Vero. Fractionation of the ethanol extract was conducted by a Vacuum Column Chromatography (VCC) and have 4 fraction that were F1, F2, F3 and F4. Cytotoxicity and cell proliferation inhibitory were tested of fraction F1, F2, F3 and F4 against cell lines of T47D. The values IC50 of F1; F2; F3 and F4 against cell lines of T47D were 82.744; 163.679; 66.522 and 333.026 μg/mL and fraction F3 concentration 31.5 µg/mL inhibits cell proliferation cell lines of T47D at 48 hours of incubation

Synthesis and antiproliferative activity of 3-(2-chloroethyl)-5-methyl-6-phenyl-8-(trifluoromethyl)-5,6-dihydropyrazolo[3,4-f][1,2,3,5]tetrazepin-4-(3H)-one

Based on the encouraging results found for 3,5-dimethyl-6-phenyl-8-(trifluoromethyl)-5,6-dihydropyrazolo[3,4-f][1,2,3,5]tetrazepin-4-(3H)-one 7 previously tested by us, as well as the consideration that heterocycle fused tetrazepinones bearing the 2-chloroethyl substituent show a better cytotoxic profile than temozolomide and mitozolomide against human cancer cell lines which express the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT), in this paper we report the multistep synthesis and the biological study of 3-(2-chloroethyl)-5-methyl-6-phenyl-8-(trifluoromethyl)-5,6-dihydropyrazolo[3,4-f][1,2,3,5]tetrazepin-4-(3H)-one 10. Like compound 7, it was active on P-glycoprotein expressing cells (MDR) HL60 and on K562 cell line that are resistant to apoptosis induced by different stimuli, showing GI50 values of 14 and 18 μM respectively. As an antiproliferative agent against the above cells compound 10 was about 2.2 times more active than compound 7. Compound 10 was also tested against WiDR cells which are overexpressing the DNA repair protein MGMT, showing a GI50 value of 2.3 μM. Finally, concerning the effect on cell cycle we observed an evident difference between compounds 7 and 10. In fact, compound 7 induces a block of cell cycle in G0-G1, therefore acting as phase-specific drug, in contrast, compound 10 is a not phase-specific agent. Both the compounds are able to increase the apoptotic sub G0-G1 peak of cell cycle.

Monocarboxylate transporter 1 inhibitors as potential anticancer agents.

Potent monocarboxylate transporter 1 inhibitors (MCT1) have been developed based on α-cyano-4-hydroxycinnamic acid template. Structure-activity relationship studies demonstrate that the introduction of p-N, N-dialkyl/diaryl, and o-methoxy groups into cyanocinnamic acid has maximal MCT1 inhibitory activity. Systemic toxicity studies in healthy ICR mice with few potent MCT1 inhibitors indicate normal body weight gains in treated animals. In vivo tumor growth inhibition studies in colorectal adenocarcinoma (WiDr cell line) in nude mice xenograft models establish that compound 27 exhibits single agent activity in inhibiting the tumor growth.

Light-controlled endosomal escape of the novel CD133-targeting immunotoxin AC133–saporin by photochemical internalization — A minimally invasive cancer stem cell-targeting strategy

The cancer stem cell (CSC) marker CD133 is an attractive target to improve antitumor therapy. We have used photochemical internalization (PCI) for the endosomal escape of the novel CD133-targeting immunotoxin AC133–saporin (PCIAC133–saporin). PCI employs an endocytic vesicle-localizing photosensitizer, which generates reactive oxygen species upon light-activation causing a rupture of the vesicle membranes and endosomal escape of entrapped drugs. Here we show that AC133–saporin co-localizes with the PCI-photosensitizer TPCS2a, which upon light exposure induces cytosolic release of AC133–saporin. PCI of picomolar levels of AC133–saporin in colorectal adenocarcinoma WiDr cells blocked cell proliferation and induced 100% inhibition of cell viability and colony forming ability at the highest light doses, whereas no cytotoxicity was obtained in the absence of light. Efficient PCI-based CD133-targeting was in addition demonstrated in the stem-cell-like, triple negative breast cancer cell line MDA-MB-231 and in the aggressive malignant melanoma cell line FEMX-1, whereas no enhanced targeting was obtained in the CD133-negative breast cancer cell line MCF-7. PCIAC133–saporin induced mainly necrosis and a minimal apoptotic response based on assessing cleavage of caspase-3 and PARP, and the TUNEL assay. PCIAC133–saporin resulted in S phase arrest and reduced LC3-II conversion compared to control treatments. Notably, co-treatment with Bafilomycin A1 and PCIAC133–saporin blocked LC3-II conversion, indicating a termination of the autophagic flux in WiDr cells. For the first time, we demonstrate laser-controlled targeting of CD133 in vivo. After only one systemic injection of AC133–saporin and TPCS2a, a strong anti-tumor response was observed after PCIAC133–saporin. The present PCI-based endosomal escape technology represents a minimally invasive strategy for spatio-temporal, light-controlled targeting of CD133+ cells in localized primary tumors or metastasis.

Design, Synthesis, and In Vitro Evaluation of Novel 3, 7-Disubstituted Coumarin Derivatives as Potent Anticancer Agents.

Twenty-seven 3, 7-disubstituted coumarin derivatives were designed, synthesized, and evaluated in vitro as anticancer agents. Most of the compounds showed moderate-to-potent antiproliferative activity against K562 cells. Compounds 7b and 7d were chosen to evaluate the concentration of 50% growth inhibition (GI50 ) against SN12C, OVCAR, BxPC-3, KATO-III, T24, SNU-1, WiDr, HeLa, K562, and AGS cell lines. The most potent compound 7d was selected for further cell cycle arrest assay in the AGS cell line. The in vitro data indicated that methylation of benzimidazole moiety at the 3-position of coumarin exhibited significant enhancement of anticancer activity. This study should provide important information for further modification and optimization of coumarin derivatives as anticancer agents.

Integrin αvβ6 sustains and promotes tumor invasive growth in colon cancer progression.

To detect the mechanism by which colon tumor escapes the growth constraints imposed on normal cells by cell crowding and dense pericellular matrices. An immunohistochemical study of integrin αvβ6 and matrix metalloproteinase-9 (MMP-9) was performed on tissue microarrays of 200 spots, including 100 cases of colon tumors. High immunoreactivity for αvβ6 (73.7%; 28/38) and MMP-9 (76.5%; 52/68) was observed in invasive tumor portions. Furthermore, the effects of integrin αvβ6 on tumor invasive growth in nude mice were detected. Tumor invasive growth and high expression of both αvβ6 and MMP-9 were only seen in tumors resulting from WiDr cells expressing αvβ6 in the tumorigenicity assay. Flow cytometry was applied to analyze αvβ6 expression in colon cancer WiDr and SW480 cells. The effects of cell density on αvβ6 expression and MMP-9 secretion were also detected by Biotrak MMP-9 activity assay and gelatin zymography assay. High cell density evidently enhanced αvβ6 expression and promoted MMP-9 secretion compared with low density. Integrin αvβ6 sustains and promotes tumor invasive growth in tumor progression via a self-perpetuating mechanism. Integrin ανβ6-mediated MMP-9 secretion facilitates pericellular matrix degradation at high cell density, which provides the basis of invasive growth.

5β,19-epoxycucurbitane triterpenoids from Momordica charantia and their anti-inflammatory and cytotoxic activity.

Five new 5β,19-epoxycucurbitane triterpenoids, taikugausins A-E (1-5), together with 5β,19-epoxy-25-methoxycucurbita-6,23-diene-3β,19-diol (6), have been isolated and characterized from the 70 % EtOH extract of the fresh fruits of Momordica charantia. The chemical structures of compounds 1-6 were elucidated on the basis of extensive spectroscopic analyses, especially 2D NMR (HMQC, HMBC, and NOESY) experiments and HRESIMS data. The relationship between NMR chemical shifts and the configuration of C-19 with an OMe group in 5β,19-epoxycucurbitane are described. Among them, compounds 3 and 4 exhibited remarkable anti-inflammatory activities by the inhibition of nitric oxide production at the concentration of 10 µg/mL. In addition, 3 and 4 also showed moderate cytotoxicity against WiDr, Hep G2, MCF-7, and HEp-2 human tumor cell lines.

Abstract 4051: Prion protein and its ligand STI1/HOP modulate migration and invasion of cell lines derived from colorectal tumors

Abstract The colorectal cancer is the fourth most common type of cancer in the world. The search for potential molecular targets for therapy has led to the Cellular Prion Protein (PrPC) as a possible candidate. Its activity depends on the interaction with the secreted form of the stress inducible protein 1 or heat shock organizing protein (STI1/HOP). Previous results indicated the importance of the PrPC-STI1/HOP complex in proliferation of glioblastomas and invasion of melanomas. Therefore, this work aims to understand how PrPC-STI1/HOP complex can influence the migration and invasion of WiDr and HCT8 colorectal tumor cells. PrPC is equally expressed at the cell surface of both cell lines. Cell migration assays were performed using boyden chambers and recombinant STI1/HOP as chemical attractant. For invasion experiments cells were seeded on boyden chambers covered with matrigel and treated with STI1/HOP. The results showed that when stimulated with recombinant STI1/HOP, both tumor cell lines showed higher migration and invasion potentials than non-treated cells or those treated with a STI1/HOP deletion mutant for the PrPC binding domain (STI1Δ230-245). Moreover, migration and invasion mediated by PrPC-STI1/HOP complex were blocked by a STI-1/HOP (230-245) peptide, which mimics STI1 binding site to PrPC or also by a neutralizing anti-PrPC antibody. STI1/HOP is secreted in the conditioned medium (CM) within extracellular microvesicles (MVs) and WiDr cells secreted more MVs and STI1/HOP than HCT8 cells. Remarkable, HCT8 cells treated with CM or isolated MVs from WiDr cells show a higher migration than that induced when the former cells were treated with their own CM or when WiDr cells were treated with CM or MVs derived from HCT8 cells. Together, these results indicate that the PrPC-STI1/HOP complex mediate migration and invasion of colorectal tumor cells and suggest that cellular migration is dependent on the concentration of secreted MVs. The role of STI1/HOP present in MVs on cell migration is still under investigation. Citation Format: Tonielli S. Lacerda, Marcos Vinicios S. Dias, Antuani Rafael Baptistella, Fernanda S. Giudice, Bruna R. Roz, Iara S. Rodrigues, Vilma Regina Martins. Prion protein and its ligand STI1/HOP modulate migration and invasion of cell lines derived from colorectal tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4051. doi:10.1158/1538-7445.AM2014-4051

Abstract 3942: Cell death resistance promoted by radiotherapy in a colorectal cell line

Abstract Colorectal adenocarcinoma is the fourth more frequent cause of death by cancer in the world. Despite some similarities, the rectal cancer has characteristics that differentiate it from the colon cancer, resulting in different treatments and outcomes. The rectal cancer in stages II and III is treated with neoadjuvant chemoradiation followed by surgery, and the response to neoadjuvant treatment in patients is variable and unpredictable. Up to now, surgery intervention is required to assess the pathologic response to therapy. The identification of predictive biomarkers for response to therapy is of great importance, in order to identify the patients that will benefit from such treatment. To better understand the molecular alterations in cells that resist to the radiotherapy, we started our research using WiDr, a colon tumor cell line. Cell cycle synchronized WiDr cells were submitted to two cycles of irradiation (4Gy in each cycle and 2 week of interval between the cycles) and resistant cells were isolated. Irradiated WiDr cells (RxTWiDr) shown a better capacity to form colonies, in colony growth assays, when compared to parental WiDr cells. This characteristic was maintained when they were submitted to a new round of irradiation. In addition, WiDrRxT cells treated with Staurosporine presented a higher viability in MTT assays than WiDr cells. We also collected the conditioned medium (CM) from RxTWiDr and WiDr cells, and treated the parental WiDr cells. The parental cells treated with CM from RxTWiDr cells for 15 minutes shown an increase in the ERK1/2 activation when compared to cells treated CM from WiDr cells. These results demonstrated that radio resistant cells are more resilient than parental cells and that their secreted factors activated specific signaling pathways via ERK1/2 phosphorylation. The nature of these factors is under investigation using proteomics. Supported by São Paulo State Foundation (FAPESP) Citation Format: Antuani R. Baptistella, Bruna Rodrigues, Marcos Dias, Fernanda Giudice, Tonielli Lacerda, Tiago Santos, Felipe Silva, Petrus Paulo Silva, Samuel Aguiar, Vilma Martins. Cell death resistance promoted by radiotherapy in a colorectal cell line. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3942. doi:10.1158/1538-7445.AM2014-3942

Antiproliferative Evaluation of N-sulfonyl-2-alkyl-six Membered Azacycles. A QSAR Study

A series of functionalized N-sulfonyl-piperidines and N-sulfonyl-tetrahydropyridines were evaluated for their antiproliferative activity against the representative panel of human solid tumor cells A2780 (ovarian), SW1573 (non-small cell lung) and WiDr (colon). The SAR study showed for WiDr cells a correlation between the biological activity and the length of the N-sulfonyl group, the nature of the substituents and the type of alkyl side chain. Further QSAR studies indicate that the size and nature of the N-sulfonyl group, the atomic polarizability (MP) and the partition coefficient are the most important descriptors for the activity. The major contribution is the size (F05C-S) of the N-sulfonyl group.

Low expression levels of microRNA-124-5p correlated with poor prognosis in colorectal cancer via targeting of SMC4.

A component of polycomb repressor complex 2, enhancer of zeste homolog 2 (EZH2), plays an important role in tumor malignancy and metastasis, while milk fat globule-epidermal growth factor-factor 8 (MFGE8) plays a key role in tumor progression and prognosis. MicroRNAs (miRs) are also critically involved in various physiological and pathological processes. We here evaluated the relationship between overall survival (OS) in colorectal cancer patients and the expression of onco-miRs and miRs, which may target EZH2 and MFGE8. Plasma and formalin-fixed paraffin-embedded (FFPE) samples were obtained from 71 colorectal cancer patients. The expression levels of miRs complementary to EZH2 and MFGE8 mRNA and cancer malignancies were evaluated. The miRs analyzed were as follows: miR-16, miR-21, miR-26a, miR-34a, miR-98, miR-101-3p, miR-101-5p, miR-124-5p (also known as miR-124*), miR-126-3p, miR-126-5p, miR-210, miR-217, and miR-630. The plasma expression levels of MFGE8 in completely resected patients were significantly lower than those in unresectable patients. Lower miR-26a expression levels were correlated with a higher probability of OS. Higher miR-124-5p expression levels in plasma and FFPE samples were correlated with a higher probability of OS. The transfection of mimic miR-124-5p into WiDr and COLO201 cells inhibited the expression of structural maintenance of chromosomes 4 (SMC4) mRNA. Our results indicate that miR-124-5p may target the tumorigenesis gene, SMC4, which suggests that expression levels of miR-124-5p in plasma and FFPE samples; therefore, the expression of MFGE8, miR-26a, and miR-124-5p in plasma may be used as biomarkers to determine the prognosis of colorectal cancer patients.

Antimicrobial and cytotoxic activities of endophytic fungi isolated from Piper crocatum Ruiz &amp; Pav

To investigate antimicrobial and cytotoxic activities of endophytic fungi isolated from Piper crocatum Ruiz & Pav (P. crocatum). Ethyl acetate extracts were obtained by liquid-liquid partition of fermentation broth of endophytes followed by evaporation. The antimicrobial activities were determined by diffusion techniques followed by thin layer chromatography-bioautography. Cytotoxicity studies were conducted using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. Data generated were used to plot a dose-response curve, of which the concentration of extract required to kill 50% of cell population (IC50) was determined. Two endophytes were isolated from leaves and stem of P. crocatum, designated as DS1 and BS1. Ethyl acetate extracts of BS1 was found to inhibit the growth of Bacillus subtilis, Escherichia coli and Staphylococcus aureus at minimum dose of 31.25, 125 and 250 µg, respectively. thin layer chromatography-bioautography of this extract resulted in at least two inhibition zones. BS1 extract also inhibited the growth of WiDr and T47D cell lines with IC50 of 120.38 µg/mL and 37.43 µg/mL, respectively. BS1 extract produced by endophytic fungi of P. crocatum is potential to be developed as source of novel bioactive compounds.

Role of N‐acetylglucosaminyltransferase‐V and galectin‐3 binding protein in anoikis stress of cancer cells (788.1)

Anoikis is an anchorage‐dependent cell death. Anoikis resistance is known to be a crucial step for cancer metastasis. We have previously identified that N‐Acetylglucosaminyl transfer ase‐V (GnT‐V) is a key protein inducing metastasis in human colon cancer cells. And the secretion of galectin‐3 binding protein (LGALS3BP) was lowered in GnT‐V overexpressing WiDr cells. In this study, we hypothesized that GnT‐V has an inhibitory effect on anoikis of cancer cells, while LGALS3BP stimulates the apoptotic process. To prove this, we established the stable transfectants (WiDr:GnT‐V), the control cells (WiDr:mock). As a result, GnT‐V was observed to mitigate anoikis and promote cell aggregation in soft‐ agar plate system. Among human colon cancer cells tested, HCT116 cells formed aggregates poorly and thus showed low cell viability. Of note, the secretion of LGALS3BP was higher than any other colon cancer cellline, which is in line with the result that anoikis resistance was observed in LGALS3BP‐suppressed WiDr cells. Taken together, these results may suggest that GnT‐V induces anoikis resistance of cancer cells either alone or in cooperation with the target molecules, such as LGALS3BP.

Up-regulation of c-Cbl suggests its potential role as oncogene in primary colorectal cancer

Dear Editor: Although progressive advances in early detection has been carried out in the last years, colorectal cancer (CRC) still represents the third cause of cancer death worldwide. This is because more than half of the patients progress to metastatic disease, which determines a very poor outcome. Therefore, it remains necessary to better understand the molecular basis that governs the progression from the early stages of CRC to metastatic development. In this context, the acquisition of invasiveness properties is a key molecular event that determines the progression from primary tumor to metastasis. Of importance, it has been recently reported that c-Cbl, an E3 ubuquitin ligase and a multifunctional adaptor protein, regulates invasion ability of cancer cells through matrix metalloproteinase 2. It is an unexpected observation since c-Cbl was largely reported to play a tumor suppressor role in several tumor types. However, in concordance with this novelproposed oncogenic role as regulator of cell adhesion, migration, and degradation, c-Cbl has been described as a marker of poor clinical outcome in prostate cancer. Therefore, it seems that this protein could be playing a dual role as a tumor suppressor or oncogene depending on the cancer model and stage of the disease. This novel-reported function for c-Cbl and the fact that its role in CRC remains unclear prompted us to analyze c-Cbl expression by western blot in a series of seven CRC cell lines. Interestingly, we observed increased c-Cbl levels in the DLD-1, RKO, LoVo, and SW620 cell lines whereas the WiDr, SW480, and HT-29 cell lines showed a c-Cbl expression similar to normal colonic mucosa. To further evaluate the importance of in CRC, we studied c-Cbl expression levels in 22 patients with primary CRC. Primary colorectal tissues were surgical resection specimens from CRC tumors obtained from Fundacion Jimenez Diaz Biobank (BFJD, Madrid). Tumor, node, metastases (TNM) staging was classified using the 7th American Joint Committee on Cancer (AJCC) staging system for colorectal cancer. Clinical data were collected from medical clinical records by an oncologist (J. G.-F.). Paired normal mucosa obtained from each patient was used as control. Moreover, a pathologist confirmed that primary tumor tissues used in this work contained greater than 70 % tumoral component (F. R.). All samples were taken anonymously and the ethical committee and institutional review board approved the project. Importantly, we found that 8 out of the 22 CRC cases showed c-Cbl overexpression in the tumor samples compared to their paired normal colonic mucosa. These results confirmed the previous observations made in CRC cell lines. Altogether, our findings suggest that c-Cbl deregulation is a recurrent event that could be playing a role in the acquisition of invasiveness properties of CRC cells and might serve as a novel potential therapeutic target in a subset of CRC patients. However, further studies are needed to clarify this potential role of c-Cbl as oncogene in primary colorectal cancer and to determine its biological and clinical significance in the pathogenesis of this disease.

Selective Cytotoxicity evaluation in Anticancer drug screening of Boehmeria virgata (Forst) Guill leaves to several human cell lines: HeLa, WiDr, T47D and Vero

Cancer is one of the leading causes of death worldwide. Many researchers have attempted to develop new treatments that will improve the prognosis of cancer patients. Indonesian forests, one of the most important world’s biodiversity, but little is known about chemical and/or pharmacological potential of these plants. The aim of this study to investigate selective cytotoxicity of B. virgata against of various cancer cell lines. The ethanolic extract of B. virgata leaves can act against HeLa, Widr, T47D and Vero cell lines with IC50 18.991 ± 0.234, 18.925 ± 1.277, 12.732 ± 0.945 and 16.022±0.663 ?g/ml with selective index 0.844, 0.847, 1.258 and 1.000, respectively. We highlight the significant cytotoxic effect of B. virgata from the leaves, which introduces promising expectations for new projects in chemistry, pharmacology and toxicology although non selective in these cell lines. Dhaka Univ. J. Pharm. Sci. 12(2): 123-126, 2013 (December) DOI: http://dx.doi.org/10.3329/dujps.v12i2.17615

Targeted and ultrasound-triggered drug delivery using liposomes co-modified with cancer cell-targeting aptamers and a thermosensitive polymer

In this study, we demonstrated the feasibility of targeted and ultrasound-triggered drug delivery using liposomes co-modified with single stranded DNA aptamers that recognized platelet-derived growth factor receptors (PDGFRs) as targeting ligands for breast cancer cells and poly(NIPMAM-co-NIPAM) as the thermosensitive polymer (TSP) to sensitize these liposomes to high temperature. TSP-modified liposomes (TSP liposomes) released encapsulated calcein under 1MHz ultrasound irradiation for 30s at 0.5W/cm2 as well as the case under incubation for 5min at 42°C. Ultrasound-triggered calcein release from TSP liposomes was due to an increased local temperature, resulting from cavitation bubble collapse induced by ultrasound, and not due to an increase in the bulk medium temperature. Liposomes modified with PDGFR aptamers (APT liposomes) bound to MDA-MB-231 human breast cancer cells through PDGFR aptamers; however, they did not bind to primary human mammary epithelial cells (HMECs). The binding of APT liposomes was greatest for MDA-MB-231 cells, followed by MCF-7, WiDr, and HepG2 cancer cells. In a cell injury assay using doxorubicin (DOX)-loaded APT/TSP liposomes and ultrasound irradiation, cell viability of MDA-MB-231 at 24h after ultrasound irradiation (1MHz for 30s at 0.5W/cm2) with DOX-loaded APT/TSP liposomes was 60%, which was lower than that with ultrasound irradiation and DOX-loaded TSP liposomes or with DOX-loaded APT/TSP liposomes alone.

Hyperthermic intraperitoneal chemotherapy using a combination of mitomycin C,5-fluorouracil, and oxaliplatin in patients at high risk of colorectal peritoneal metastasis: A Phase I clinical study

The drugs and protocols used for hyperthermic intraperitoneal chemotherapy (HIPEC) vary among institutions. Here we show the efficacy of the 3-drug combination of mitomycin C (MMC), 5-fluorouracil (5FU), and oxaliplatin (OHP) in an in vitro simulation of HIPEC and the safety of HIPEC with these drugs during a Phase I study of patients at high risk of developing colorectal peritoneal metastasis. To simulate HIPEC, we used HCT116 and WiDr cells to assess the growth inhibitory efficacy of MMC 2 μg/mL, 5FU 200 μg/mL, and OHP 40 μg/mL as single drugs or their combination after an exposure time of 30 min at 37 or 42 °C. In addition, nine patients underwent surgical resection of tumors and HIPEC with MMC, 5FU, and an escalating dose of OHP (90/110/130 mg/m²). Dose-limiting toxicity was monitored. In the simulation, the 3-drug combination showed marked tumor-suppressive effects compared with those from ten times higher dose of OHP 400 μg/mL, with significant augmentation under hyperthermic conditions. No dose-limiting toxicity occurred in the clinical study. Dose escalation was completed at the final level of OHP. The MMC-5FU-OHP combination showed marked growth inhibition against colorectal cancer cells under hyperthermic conditions in vitro. In the phase I study, the recommended dose of OHP was determined as 130 mg/m² when used with MMC and 5FU; HIPEC using MMC-5FU-OHP appears to be safe and feasible for patients at high risk of colorectal peritoneal metastasis.

Aberrant SERCA3 expression during the colorectal adenoma-adenocarcinoma sequence

Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 3 is involved in calcium mobilization from the endoplasmic reticulum into the cytosol and is closely linked to metabolism, neuronal plasticity, gene transcription, cell growth, differentiation, apoptosis, protein folding and carcinogenesis. In order to elucidate the role of SERCA3 in colorectal carcinogenesis and subsequent progression, its expression was examined using immunohistochemistry and in situ hybridization (ISH) on tissue microarrays containing colorectal carcinomas, adjacent non-neoplastic mucosa (NNM) and adenoma, and metastatic carcinoma in lymph node and liver. Colorectal carcinoma tissue and cell lines were assessed for SERCA3 expression by western blotting or RT-PCR, respectively. SERCA3 was distinctively expressed in Colo201, Colo205, DLD-1, HCT-15, HCT-116, HT-29, KM-12, SW480, SW620 and WiDr cells at both the mRNA and protein levels. SERCA3 mRNA expression was low in carcinoma when compared to that in matched NNM by quantitative PCR (P<0.05), while the converse was true by ISH. Lower expression of SERCA3 was immunohistochemically observed in colorectal carcinoma when compared to that in NNM and adenoma (P<0.05). In contrast, primary carcinoma showed high SERCA3 expression when compared to that in metastatic carcinoma in lymph node or liver by IHC (P<0.05). Immunohistochemically, SERCA3 expression was negatively related to lymphatic invasion, but not with age, gender, depth of invasion, venous invasion, lymph node metastasis, distant metastasis, TNM stage, degree of differentiation or survival rate (P>0.05). There was a positive relationship between SERCA3 expression and serum CEA levels in the carcinoma patients (P<0.05). Cox's proportional hazards model indicated that depth of invasion and distant metastasis are independent prognostic factors for overall colorectal carcinomas (P<0.05). These findings suggest that aberrant SERCA3 expression is closely linked to the adenoma-adenocarcinoma sequence and progression of colorectal carcinomas.

Comparison of the effects of photon versus carbon ion irradiation when combined with chemotherapy in vitro

BackgroundCharacterization of combination effects of chemotherapy drugs with carbon ions in comparison to photons in vitro.MethodsThe human colon adenocarcinoma cell line WiDr was tested for combinations with camptothecin, cisplatin, gemcitabine and paclitaxel. In addition three other human tumour cell lines (A549: lung, LN-229: glioblastoma, PANC-1: pancreas) were tested for the combination with camptothecin. Cells were irradiated with photon doses of 2, 4, 6 and 8 Gy or carbon ion doses of 0.5, 1, 2 and 3 Gy. Cell survival was assessed using the clonogenic growth assay. Treatment dependent changes in cell cycle distribution (up to 12 hours post-treatment) were measured by FACS analysis after propidium-iodide staining. Apoptosis was monitored for up to 36 hours post-treatment by Nicoletti-assay (with qualitative verification using DAPI staining).ResultsAll cell lines exhibited the well-known increase of killing efficacy per unit dose of carbon ion exposure, with relative biological efficiencies at 10% survival (RBE10) ranging from 2.3 to 3.7 for the different cell lines. In combination with chemotherapy additive toxicity was the prevailing effect. Only in combination with gemcitabine or cisplatin (WiDr) or camptothecin (all cell lines) the photon sensitivity was slightly enhanced, whereas purely independent toxicities were found with the carbon ion irradiation, in all cases. Radiation-induced cell cycle changes displayed the generally observed dose-dependent G2-arrest with little effect on S-phase fraction for all cell lines for photons and for carbon ions. Only paclitaxel showed a significant induction of apoptosis in WiDr cell line but independent of the used radiation quality.ConclusionsCombined effects of different chemotherapeutics with photons or with carbon ions do neither display qualitative nor substantial quantitative differences. Small radiosensitizing effects, when observed with photons are decreased with carbon ions. The data support the idea that a radiochemotherapy with common drugs and carbon ion irradiation might be as feasible as respective photon-based protocols. The present data serve as an important radiobiological basis for further combination experiments, as well as clinical studies on combination treatments.