An overview of yeast probiotics as cancer biotherapeutics: possible clinical application in colorectal cancer.

The previous reports have established a strong link between diet, lifestyle, and gut microbiota population with the onset of the colorectal cancer (CRC). Administration of probiotics has become a particular interest in prevention and treatment of CRC. As potential dietary complements, probiotics might be able to lower the risk of CRC and manage the safety of traditional cancer therapies such as surgery, radiation therapy, and chemotherapy. This review investigates the promising effects of probiotics as biotherapeutics, with due attention to possible clinical application of yeast probiotics in prevention and treatment of CRC. In addition, various underlying anti-cancer mechanisms are covered here based on scientific evidence and findings from numerous experimental studies. Application of probiotics as biotherapeutics in CRC, however, needs to be approved by human clinical trials. It is of prime concern, to find potential probiotic strains, effective doses for administrations and regimes, and molecular mechanisms involved in prevention and treatment.

Optimizing expression of antiviral cyanovirin-N homology gene using response surface methodology and protein structure prediction.

Cyanovirin-N (CVN) is well known as an anti-HIV protein. The efficient production of low cost microbicides for preventing the HIV-infection  has lately become a requirement worldwide. The aim of the present study was to optimize the expression of antiviral Cyanovirin-N homology gene found in the indigenous strain of Nostoc ellipsospourum LZN using Response Surface Methodology (RSM) and Protein Structure Analysis. Optimization of three induction factors (IPTG concentration (0.1, 0.55 and 1mM), temperature for bacterial growth (20, 28.5 and 37°C) and induction time (4, 10 and 16h) was done using RSM and Box-Behnken Design. Total RNA extraction was performed and mRNA levels were quantified in each experimental design by one-step SYBR qPCR. Protein structure was predicted using I-TASSER server. The full-length sequence of LZN-CVN gene is 306 bp in length, due mostly to five mutations. RSM analysis showed that the optimum condition to obtain maximum fold change was a concentration of 0.6mM IPTG, temperature set to 29°C and a 12h long induction time. The extracted protein from periplasmic fraction (8 kDa) was verified via SDS-PAGE. The high percentage of LZN-CVN similarity was demonstrated with PDB (Protein Data Bank) accession code of 2rp3A (CVN domain B mutant) and the ligand binding sites were related to N42, V43, D44, G45, S52, N53 and E56 residues. Different expression systems could assist in the development of anti-HIV proteins in a large scale. The LZN-CVN protein was successfully expressed in the E.coli system. RSM could be applied to a series of mathematical and statistical methods for modeling and analysis of responses which are influenced by various variables of interest.

Co-inhibition of Notch and NF-κB Signaling Pathway Decreases Proliferation through Downregulating IκB-α and Hes-1 Expression in Human Ovarian Cancer OVCAR-3 Cells.

Background: Ovarian cancer is one of the most lethal gynecological malignancies and numerous changes in signaling cascades are involved in the initiation and progression of ovarian cancerous cells. Here, we investigated the role of NF-κB and Notch pathways inhibition on human ovarian cancer OVCAR-3 cells proliferation and IκB-α and Hes-1 expression as 2 key genes in these pathways regulation. Methods: The effects of Bay 11-7085 and DAPT, NF-κB and Notch pathways specific inhibitors, on cell proliferation were evaluated using MTT assay. In addition, the cells were transfected by Notch and IKK-ß siRNAs. mRNA and protein levels of target genes were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot after 48 h incubation with inhibitors and siRNAs. Results: Bay 11-7085 and DAPT significantly decreased the cell proliferation OVCAR-3. IκB-α and Hes-1 mRNA levels decreased to 5 or 3% and 6% or 2% after treatment with Bay 11-7085 or DAPT, respectively (p<0.05). We also found that combination treatment exert a more potent effects on the expression of these gene (p<0.05). Moreover, siRNA transfection caused a significant reduction in IκB-α and Hes-1 mRNA levels (p<0.05). In the protein level, OVCAR-3 cell treatment with both chemichal inhibitors and specific siRNA cause a significant decrease in the expression of target genes (p<0.05) Conclusion: Our findings suggest that inhibition of NF-κB and Notch signaling pathways can effectively reduce OVCAR-3 cells proliferation. Therefore, pharmacological targeting of the NF-κB and Notch signaling pathway could be a promising future treatment of ovarian cancer.

Downregulation of Notch Signaling Pathway as an Effective Chemosensitizer for Cancer Treatment.

Despite remarkable progress in cancer treatment, development of drug resistance is still a big burden to eliminate all tumor cells and a mean cause for tumor recurrence. Recent studies have been revealed the contribution of many signaling pathways in acquisition of resistance to chemotherapy. Because of its potential in maintaining the balance between cell proliferation and apoptosis, Notch signaling pathway has mean relevance to various aspects of cancer biology, from cancer stem cells to tumor immunity to multidrug resistance. Therefore, Notch signaling pathway is an attractive target for cancer therapy because targeting Notch signaling could overcome multi drug resistance (MDR). This article will provide a brief overview of the published evidences in support of Notch targeting in reverting multidrug resistance as a safer and novel approach for the improvement of tumor treatment.

PPAR-gamma in overcoming kinase resistance in chronic myeloid leukemia.

Peroxisome proliferator-activated receptor gamma (PPARγ) plays key roles in regulating cellular differentiation, proliferation and apoptosis pathways. As such, they are considered promising targets for anticancer drug development, especially for breast cancer, multiple myeloma and hematologic malignancies. Chronic myeloid leukemia (CML) is a myeloproliferative disorder arising from an oncogenic Bcr-Abl tyrosine kinase. Inhibitors of this oncogene by small molecules such as imatinib are effective only in 75% of the patient's population. One of the potential strategies to overcome this resistance is to devise combination therapy protocols with other therapeutic agents including PPAR ligands. Since PPAR ligands are potentially interesting in different hematologic malignancies, this article will review the potential of PPAR ligands for use in CML treatment.

RESISTIN EFFECT ON TELOMERASE GENE EXPRESSION IN GASTRIC CANCER CELL LINE AGS.

BACKGROUND: Resistin, as an adipokine, has been shown to be increased in serum plasma of gastric cancer patients and suggested to be a major factor in gastric carcinogenesis. However, it is still not clear how Resistin influences gastric cancer progression. The aim of this study was to evaluate Resistin effect on cell proliferation and expression of telomerase gene in gastric cancer cell line (AGS). METHODS: In this study, the proliferating activity of AGS cells stimulated with Resistin was also evaluated by using 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT) assay and trypan blue staining method. To investigate telomerase gene expression affected by Resistin, total RNA was extracted, cDNA was synthesized and expression of hTERT mRNA was carried out by real-time reverse transcription polymerase chain reaction. RESULTS: Exogenous Resistin has induced gastric cancer cells proliferation in a dose-dependent manner and could improve cell viability. Also the expression of Human Telomerase Reverse Transcriptase (hTERT) was upregulated in 24 hours, after Resistin treatment. CONCLUSIONS: This study has shown Resistin induces exogenously gastric cancer cell proliferation and increases hTERT gene expression. These findings may clarify the role of Resistin in gastric carcinogenesis. Therefore blocking Resistin signaling and limiting its secretion may be valuable for the treatment of gastric cancer.

Differential effects of peroxisome proliferator-activated receptor agonists on doxorubicin-resistant human myelogenous leukemia (K562/DOX) cells.

P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in tumor cells is still a main obstacle for the chemotherapeutic treatment of cancers. Therefore, identification of safe and effective MDR reversing compounds with minimal adverse side effects is an important approach in the cancer treatment. Studies show that peroxisome proliferator-activated receptor (PPARs) ligands can inhibit cell growth in many cancers. Here, we investigated the effect of different PPAR agonists include fenofibrate, troglitazone and aleglitazar on doxorubicin-resistant human myelogenous leukemia (K562/DOX) cells. The effects of doxorubicin (DOX) following treatment with PPAR agonists on cell viability were evaluated using MTT assay and the reversal fold (RF) values. Rhodamine123 (Rh123) assays were used to determine P-gp functioning. P-gp mRNA/protein expression was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analysis after incubation with troglitazone and aleglitazar. Our results showed that troglitazone and aleglitazar significantly enhanced the cytotoxicity of DOX and decreased the RF values in K562/DOX cells, however, no such results were found for fenofibrate. Troglitazone and aleglitazar significantly down regulated P-gp expression in K562/DOX cells; in addition, the present study revealed that aleglitazar elevated intracellular accumulation of Rh123in K562/DOX cells as short-term effects, which also contribute to the reversal of MDR. These findings show that troglitazone and especially aleglitazar exhibited potent effects in the reversal of P-gp-mediated MDR, suggesting that these compounds may be effective for combination therapy strategies and circumventing MDR in K562/DOX cells to other conventional chemotherapeutic drugs.

Visfatin effects on telomerase gene expression in AGS gastric cancer cell line.

AIM: The aim of this study was to assess visfatin expression and its effect on human telomerase gene expression in AGS gastric cancer cell line. MATERIALS AND METHODS: In this study, human gastric cancer (AGS) cell line was established as an in vitro model. Reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay was performed to show that visfatin expression in messenger ribonucleic acid (mRNA) and protein level respectively. After stimulating with increasing concentrations of visfatin for times of 24 h and 48 h, cell proliferation was assessed by 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT) assay. In order to investigate telomerase gene expression affected by visfatin in AGS cell line, total RNA was extracted and complementary deoxyribonucleic acid was synthesized buy using commercially available kits. Expression of human telomerase reverse transcriptase (hTERT) mRNA was carried out by real-time RT-PCR. RESULTS: After visfatin treatment gastric cell line proliferation was enhanced and was increased the expression of hTERT. CONCLUSIONS: The obtained data showed that visfatin induces endogenously gastric cancer cell proliferation and increases telomerase (hTERT) gene expression, as a cancer gene. Based on this study, it is suggested that expression of this adipocytokine protein in real samples could be biomarker for gastric cancer.

Development and Evaluation of a Cetuximab-based Humanized Single Chain Antibody Against EGFR-overexpressing Tumors.

Production of humanized single chain antibodies (hscFv) can potentially be a powerful solution to limitations imposed by large size and murine nature of cetuximab. The present study describes generation of a cetuximab-based hscFv using CDR-grafting method. Cetuximab CDRs were grafted on frameworks selected from human germline antibody sequence repertoire. The strategy employed in selecting human sequences was the highest sequence similarity of variable domains between human and parental antibodies as well as similarity in the CDRs canonical structures. To maintain the binding affinity, the parental vernier zone residues were retained murine in hscFv. Recombinant hscFv was expressed in E. coli and affinity purified by Ni-NTA column. The potency of hscFv in targeting EGFR was evaluated using A431, a cell line over-expressing EGFR. Dot blot and ELISA tests were used to assess the reactivity and MTT assay to evaluate the growth inhibition of hscFv on A431 cell line. The humanization of cetuximab variable regions resulted in 22.2% increase in humanness of hscFv. Reactivity analyses of hscFv on A431 cells showed better binding affinity and higher growth inhibition effect (2.6 times) comparing to murine counterpart. In conclusion, hscFv produced in this study displayed reduced potential immunogenicity as well as enhanced cytotoxic effect on EGFR- overexpressing tumor cells.

The Possible Impact of Obesity on Androgen, Progesterone and Estrogen Receptors (ERα and ERß) Gene Expression in Breast Cancer Patients.

BACKGROUND: Obesity has been associated with increased mortality from hormone dependant cancers such as breast cancer which is the most prevalent cancer in women. The link between obesity and breast cancer can be attributed to excess estrogen produced through aromatization in adipose tissue. The role of steroid hormone receptors in breast cancer development is well studied but how obesity can affect the expression pattern of steroid hormones in patients with different grades of breast cancer was the aim of this study. METHODS: In this case-control study, 70 women with breast cancer participated with different grades of obesity (36 none obese, BMI < 25 kg/m(2) and 34 obese, BMI ≥ 25 kg/m(2)). The mean age of participants was 44.53 ± 1.79 yr (21-70 yr). The serum level of estrogen, progesterone and androgen determined by ELISA. Following quantitative expression of steroid hormone receptors mRNA in tumor tissues evaluated by Real-time PCR. Patients with previous history of radiotherapy or chemotherapy were excluded. SPSS 16 was used for data analysis and P < 0.05 considered statistically significant. RESULTS: The difference in ERα, ERß and PR mRNA level between normal and obese patients was significant (P < 0.001). In addition, the expression of AR mRNA was found to be higher than other steroid receptors. There was no significant relation between ERß gene expression in two groups (P = 0.68). We observed a significant relationship between ERα and AR mRNA with tumor stage and tumor grade, respectively (P = 0.023, P = 0.015). CONCLUSION: According to the obtained results, it is speculated that obesity could paly a significant role in estrogen receptors gene expression and also could affect progression and proliferation of breast cancer cells.

Prostate-specific antigen gene expression and telomerase activity in breast cancer patients: possible relationship to steroid hormone receptors.

Breast cancer, the most prevalent cancer among women, is a steroid hormone receptor-dependent cancer. Recently, it has been shown that telomerase and prostate-specific antigen (PSA) gene expressions are under control of steroid hormone receptors. The aim of this study was to investigate the relationship between telomerase activity and PSA gene expression with steroid hormone receptors in breast cancer patients. This study consisted of 50 women with breast benign tumors and 50 malignant (invasive) tumors. Telomerase activity was measured in tumor cytosol of samples by telomeric repeat amplification protocol (TRAP) assay. PSA protein and its mRNA expression were measured using ultrasensitive immunoassay and RT-PCR technique in all tumor tissues, respectively. Estrogen and progesterone receptors were stained using immunohistochemistry in tumor tissues. Telomerase activity was detected in all of the invasive breast cancer tissues. The difference of relative telomerase activity (RTA) values between stages and grades were statistically significant (p < 0.05). The PSA mRNA was detected only in benign tumors and stage I and grade I malignant tumor cytosol. Difference of tumor cytosol PSA levels between the cases and control groups and also between all grades and stages of diseases were significant (p < 0.05). There was an inverse significant correlation between the RTA and PSA protein levels in the case groups (r = -0.42, p < 0.05). There was a statistically significant difference between ER/PR with PSA level and telomerase activity in tumor tissues (p < 0.05). It is speculated that differential expression of PSA and telomerase genes in breast tumors are under control of steroid hormone receptors and could be used as a target for treatment in the future.

On-admission level of serum d-dimer and the severity of community-acquired pneumonia.

This study aims at evaluating on-admission serum level of d-dimer in patients with community-acquired pneumonia concerning the severity of the disease and in-hospital outcome of the patients. Sixty patients with community-acquired pneumonia were studied during a one-year period in Imam Khomeini and Sina Hospitals, Tabriz, Iran. On-admission serum d-dimer was measured by enzyme-linked immunoabsorbent assay and the severity of disease determined according to PORT grading system. In-hospital outcome was determined in regard to the level of serum d-dimer. Sixty patients with community-acquired pneumonia, 39 males and 21 females were enrolled. There were twelve patients with PORT one, eight patients with PORT two, eight patients with PORT three, twenty patients with PORT four and twelve patients with PORT five. The mean level of serum d-dimer was significantly higher in severe disease (p < 0.001), patients with hospital stay longer than one week (p = 0.003), patients with bronchopulmonary pattern (p = 0.012), cases in-need of mechanical ventilation (p < 0.001) and patients who expired during hospital stay (p = 0.022). On-admission level of serum d-dimer was significantly and independently higher in patients with severe disease (p < 0.001) and in cases with bronchopulmonary pattern on chest x-ray (p = 0.035). On-admission level of serum d-dimer may predict the severity of community-acquired pneumonia. Further studies are recommended for accurate cut-off points.

Prostate-specific antigen in serum during the menstrual cycle.

We previously found that prostate-specific antigen (PSA) expression in the female breast is regulated by steroid hormones and their receptors. We have now examined whether the PSA concentration in serum changes during the menstrual cycle of healthy women. Among 14 women studied, 3 had serum PSA > or = 4 ng/L; their changes in PSA content during the menstrual cycle were studied in 7 informative cycles. We found that PSA concentrations in serum are highest during the mid- to late follicular phase, drop continuously with a half-life of 3-5 days between the late follicular phase and mid-cycle, and reach a minimum during the mid- to late luteal phase. PSA changes do not correlate with changes in lutropin (LH), follitropin (FSH), or estradiol concentrations. However, PSA peaks seem to follow the progesterone concentration peaks, with a delay of 10-12 days. Sera of some volunteers were tested for their ability to upregulate PSA protein and PSA mRNA in a tissue culture system based on the T-47D breast carcinoma cell line. Only sera obtained during the mid- to late luteal phase were able to upregulate the PSA mRNA and protein. In stimulation experiments in vitro, progesterone, but not LH, FSH, estradiol, human chorionic gonadotropin, prolactin, or growth hormone, was able to upregulate PSA mRNA and protein in the T-47D cell line. These data suggest that PSA is produced in a cyclical manner during the menstrual cycle.

Frequency of expression of prostate-specific antigen mRNA in lung tumors.

The presence of prostate-specific antigen (PSA) protein and messenger RNA (mRNA) was studied in 52 primary lung tumor tissues. The PSA protein was detected more frequently and at higher levels in lung tumor extracts from men. The levels of PSA protein in tumor extracts correlated with preoperative and postoperative serum PSA levels, suggesting a possible contamination of the tumor extracts with PSA from residual blood in the tumor vasculature. The PSA mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot hybridization in 24 (68%) of 35 tumors from men, in 9 (53%) of 17 tumors from women, and in 5 (71%) of 7 adjacent normal lung tissue specimens. The levels of PSA protein did not associate with patient age, the tumor stage, grade, or histologic type, or the nodal status. Similarly, PSA mRNA was not associated with any clinicopathologic variables, but squamous cell carcinomas, especially in men, were more frequently positive. A by-product of the RT-PCR procedure was cloned and sequenced and found to be a 450-base pair sequence not previously deposited in the data bank. We conclude that PSA mRNA and protein frequently can be detected in lung tumors and normal tissues from men and women but at levels much lower than those seen in breast carcinomas in women. The significance of the new 450-base pair sequence remains to be determined.

Expression of the prostate specific antigen gene by lung tissue.

We describe a female patient with lung adenocarcinoma whose tumor extract was highly positive for prostate specific antigen (PSA) immunoreactivity. PSA was present in its Mr 33,000 free form. Using reverse transcription-PCR, we were able to amplify a 754-bp fragment that specifically hybridized to a PSA RNA probe on Southern blots. The PCR fragment was sequenced and found to represent PSA cDNA and not human glandular kallikrein cDNA. PSA immunoreactivity in the lung tissue was localized by immunohistochemistry to normal epithelial cells adjacent to the tumor which was completely negative for PSA. Tissue culture experiments suggested that beclomethasone, a glucocorticoid used to treat the patient, was able to up-regulate PSA gene expression. This is the first report that unequivocally demonstrates PSA expression in lung tissue. We speculate that PSA expression was mediated by the exogenously administered steroid beclomethasone.

Steroid hormone regulation of prostate-specific antigen gene expression in breast cancer.

We have recently reported that about 30-40% of female breast tumours produce prostate-specific antigen (PSA) and that PSA production is associated with the presence of oestrogen (ER) and progesterone (PR) receptors. We have now developed a tissue culture system to study the regulation of the PSA gene in breast cancer. The breast carcinoma cell line T-47D produces PSA when stimulated by androgens, progestins and glucocorticoids/mineralocorticoids but not oestrogens. PSA mRNA appears approximately 2 h after stimulation; PSA protein appears after 4-8 h. Among 38 compounds tested, only androgens and progestins were able to stimulate PSA production at concentrations below 10(-9) M. Evidence that the progesterone and androgen receptors can regulate the PSA gene independently was provided as follows: (a) the progestin norgestimate, which does not bind to the androgen receptor, up-regulates the PSA gene at concentrations as low as 10(-10) M; (b) triamicinolone acetonide, which does not bind to the androgen receptor (AR) but binds to the PR, acts similarly to norgestimate; (c) the antiandrogen cyproterone acetate, which blocks the androgen receptor but has progestational activity, up-regulates the PSA gene at concentrations as low as 10(-10) M; (d) the antiprogestine mifepristone completely blocks the stimulation of the specific progestin norgestimate. Our tissue culture system identified androgen-progestin agonist activities of 17 alpha-ethinyloestradiol, the antioestrogen RU56, 187 and the antiprogestin mifepristone. Our data suggest that the expression of the PSA gene in the female breast is under the control of androgens and progestins. Our tissue culture system is a highly sensitive in vitro method for evaluating the biological activity of candidate compounds having agonist and antagonist steroid hormone activity.

Detection of prostate-specific antigen mRNA and protein in breast tumors.

We have developed reverse transcription-polymerase chain reaction (RT- PCR) methods for detecting prostate-specific antigen (PSA) mRNA. Using these methods, and a highly sensitive immunofluorometric assay for measuring PSA protein, we have assessed the concentrations of PSA mRNA and PSA protein in 30 primary breast tumors and a few other control tissues. We found good agreement between presence of PSA protein and PSA mRNA in breast tumors. We thus propose that, in women, detection of PSA protein or PSA mRNA in tissues and tumors offers equivalent information. Because PSA protein is present in male blood and thus could contaminate extracts from tumors and tissues from men, we propose that the RT-PCR methods we describe be used to assess nonprostatic expression of the PSA gene in men.

Creatine kinase BB isoenzyme levels in tumour cytosols and survival of breast cancer patients.

Creatinine kinase BB (CK-BB) is elevated in many tumours including those of the breast. We have recently described a new, highly sensitive and specific method for measuring CK-BB, based on monoclonal antibodies and time-resolved fluorometry. Using this method, we quantitated CK-BB in 172 breast tumour cytosols and examined the associations between CK-BB and other clinicopathological variables and patient survival. High CK-BB levels were seen more frequently in tumours from patients who were younger (age < 50 years), patients who qualified for chemotherapy and patients with oestrogen receptor-positive tumours. No association was seen between CK-BB and tumour stage, grade, size, histological type or the progesterone receptor. In univariate analysis, the risk of relapse or death was higher in the group with tumours containing high CK-BB levels but the difference did not reach statistical significance. In multivariate analysis, the risk of death was statistically significantly higher in the high-CK-BB group. Analysis of subsets of patients revealed that patients with oestrogen receptor-negative cancer have higher risk of death if their tumours contain high levels of CK-BB. Our data suggest that, in general, CK-BB is associated with more aggressive tumours but its value as a prognostic indicator is limited. CK-BB content of breast tumours may be more useful as an aid in selecting therapy directed at inhibiting this enzyme activity and thus depriving tumour cells of their energy source.

Prognostic value of creatine kinase BB isoenzyme in epithelial ovarian carcinoma.

Creatine kinase BB isoenzyme (CK-BB) is overexpressed in many tumor tissues, including ovarian cancer. Using a highly sensitive and specific immunofluorometric method, CK-BB levels in 89 primary ovarian cancer cytosolic extracts have been measured and the associations between CK-BB and clinicopathological features of ovarian cancer have been studied. It was found that CK-BB levels are higher in endometrioid cell carcinomas. No clear association was established between CK-BB levels and patient age, menopausal status, clinical stage, histological grade or size of residual tumor. CK-BB was not associated significantly with either disease-free or overall survival of the patients. Based on these data, it was concluded that there is no prognostic value of CK-BB in ovarian cancer. Drugs that target CK-dependent energy metabolism of tumor cells may not be selective in ovarian cancer therapy.