Molecular regulation of polycystic ovary syndrome: altered gene expression levels in mouse models pretreatment and post-treatment.

Polycystic ovary syndrome (PCOS) is a complex disorder and genetic factors are believed to play a role. The main aim was to investigate expression levels of genes involved in PI3K/AKT signalling pathway pretreatment and post-treatment. Mouse models of PCOS were generated. Group one included control mice with no polycystic ovaries (n = 4), Group 2 included a PCOS mouse model (n = 8), Group 3 included PCOS mice treated with clomiphene citrate (n = 7) and Group 4 included PCOS mice treated with clomiphene citrate, metformin and pioglitazone (n = 8). Histochemical analyses were performed. Total RNA was extracted and cDNA was synthesized. Irs, Akt1 and Akt2, mTor and Pdpk1 gene expression levels were evaluated by RT-PCR amplification. In Group 1, cortex and medulla were evaluated as normal; in Group 2, ovarian cortex was composed of immature oocytes and cystic follicles with atretic follicles. In Groups 3 and 4, follicles were in the process of normal follicle differentiation. The expression levels of Akt1 and Pi3k were significantly different (P < 0.0001) between Groups 1 and 2. The significant differences in expression levels of Pi3k and Akt1 were also observed between the Group 1 and both Groups 3 and 4 (P < 0.0001). Furthermore, significant variations of the expression levels of mTor between Groups 1 and 4 were observed. The extrapolation of results of this study may imply that follicular development may be regulated by molecular pathways involving Pi3k, Akt1 and mTor expression. Therefore, genes in the PI3K/AKT pathway may have a direct regulatory role in the development of PCOS.

The effects of sildenafil on smoke induced lung inflammation in rats.

OBJECTIVE: Sildenafil is a selective and potent inhibitor of cyclic guanosine monophosphate specific phosphodiesterase-5 and has anti-inflammatory effects. The aim of the study was to evaluate the effects of sildenafil on smoke-induced lung inflammation. MATERIAL AND METHODS: Twenty-nine Wistar-Albino rats were enrolled into 3 groups as control, smoker and sildenafil groups. Smoker and sildenafil groups were exposed to cigarette smoke for 2 hours per day for 8 weeks. Sildenafil 10 mg/kg/day was administered to the sildenafil group by nasogastric lavage after smoke exposure. The degree of lung inflammation was scored histopathologically for each group. RESULTS: The inflammation score was 7.25±0.93 in the control group, 8.18±1.21 in the smoker group and 7.08±1.66 in the sildenafil group. There was a non-significant decrease of inflammation score in sildenafil group with respect to control or smoker groups. While there was no significant difference of oedema, hyperemia, hemorrhage and mononuclear cell infiltration scores among the groups, it was found that the thickness of interalveolar septum and alveolar distortion was decreased in sildenafil group. However this decrease was not statistically significant. CONCLUSION: This study suggests that sildenafil might reduce smoke-induced inflammation in rat lungs. Future studies are needed in order to investigate the clinical effectiveness of this finding in smoking related lung diseases.

A comparison of the molecular distribution of proangiogenic factors in endometrium of missed abortions and of voluntary first trimester termination cases.

OBJECTIVE: The authors aimed to evaluate the angiogenic changes that occur in the cases with missed abortions compared with the voluntary termination of pregnancy as control group, with this controlled clinical study. MATERIALS AND METHODS: The study included fifteen healthy volunteer women with unwanted pregnancy less than 10th gestational week in an academic research environment. The patients were 19 women between 6th and 11th gestational weeks diagnosed with missed abortion as the patient group. Immunohistochemistry was utilized to examine temporal and spatial expression of vascular endothelial growth factor (VEGF) and their two receptors: VEGF-R1 (Flt-1) and VEGF-R2 (Flk-1/KDR), and Trombospondin-1, eNOS, iNOS, and HIF-1α in the both deciduas and placenta of the both groups. RESULTS: This study discovered the significant difference (p < 0.005) between the groups of controlled and missed abortion in the decidual and placental cell components, and has put forward that thrombospondin and iNOS have an impact on abortion through antiangiogenic effect in cases of missed abortions. CONCLUSIONS: The potential role of molecules affecting angiogenesis in the etiology of missed abortion has been evaluated and the authors aimed for this to be a guide for studies on further treatments and on the prevention of the development of missed abortions.

The expression of Forkhead transcription factors in decidua and placenta in patients with missed abortion.

BACKGROUND: Forkhead transcription factors 3a (FOXO3a) has pleiotropic biological functions in the female reproductive tract. FOXO3a has a function in decidualization, in placental development, and also in inhibition of apoptosis. This study aims to investigate a possible role of FOXO3a in missed abortion. MATERIALS AND METHODS: Decidual and placental tissue samples were obtained from the women with unwanted pregnancy as the control group and with missed abortion as the patient group. Immunohistochemistry technique was utilized to compare FOXO3a expression of the decidual cells in uterine decidual stroma and cytotrophoblast-syncytiotrophoblast cells in placental villous stroma. Immunohistochemistry was evaluated semiquantitatively utilizing the H-score technique. Results: It was demonstrated that H-Scores of FOXO3a expression in both uterine decidual stroma were increased in the missed abortion group (255.83 +/- 12.41) than in the normal pregnancy group (133.33 +/- 17.43). It was also shown that there was no difference between non-decidual area of the endometrium of the normal pregnancy and the missed abortion group (30.33 +/- 4.32; 39.66 +/- 14.30, respectively) and placental villous stroma (13.00 +/- 1.89; 13.00 +/- 1.67, respectively). However, the immunoreactivity of cytotrophoblast and syncytiotrophoblast cells significantly increased in the missed abortion group (18.83+1.47; 322.00 +/- 6.06, respectively) than in the normal pregnancy group (11.00 +/- 1.26; 254.00 +/- 8.17, respectively) (p < 0.05). CONCLUSION: These data support the hypothesis that increased FOXO3a expression in missed abortion may prevent the discharge of dead fetus to maintain decidualization, prevention of oxidative stress, immunomodulation, and inhibition of apoptosis.

Clinical significance of serum tenascin-C levels in breast cancer.

Tenascin-C (TNC) is a key molecule in tissue remodeling, and high levels are observed in many diseases, including heart failure, thrombosis, atherosclerosis, and cancer. High TNC expression by immunohistochemical analysis has been shown in invasive and metastasizing tissues from a variety of cancers, including colon, lung, brain, and breast. This study was conducted to investigate the serum level of TNC in breast cancer patients and its relationship with tumor progression and known prognostic parameters. Ninety-six breast cancer patients were enrolled into the study. Serum samples were obtained on first admission before adjuvant and metastatic treatments were given and at follow-up. Serum TNC levels were determined by the solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) method. Median age of diagnosis was 48 years old (range, 29-80). Thirty-seven (39 %) patients had metastatic breast cancer. The mean TNC levels were found to be significantly higher in patients with breast cancer (344.1 ± 42.4 pg/mL) compared to those in healthy controls (137.2 ± 26.8 pg/mL) (p = 0.005). Serum TNC level in grade 3 tumors was found to be significantly higher than in grades 1-2 tumors (p = 0.04). No correlation was detected between serum TNC levels and other prognostic parameters analyzed, including presence of metastasis, lymph node involvement, and tumor size. Serum TNC level had no significantly adverse effect on survival in univariate and multivariate analyses (p = 0.65 and p = 0.85, respectively). In conclusion, although serum TNC levels are elevated, it has no predictive or prognostic roles on survival in breast cancer patients.

The diagnostic, predictive, and prognostic role of serum epithelial cell adhesion molecule (EpCAM) and vascular cell adhesion molecule-1 (VCAM-1) levels in breast cancer.

The purpose of this study was to determine the clinical significance of vascular cell adhesion molecule-1 (VCAM-1) and epithelial cell adhesion molecule (EpCAM) in breast cancer (BC) patients. Ninety-six BC patients and 30 age- and sex-matched healthy controls were enrolled into this study. Pretreatment serum markers were determined by the solid-phase sandwich (enzyme-linked immunosorbent assay (ELISA)). The median age at diagnosis was 48 years (range 29-80 years). Majority of the patients (71 %) had luminal subtype, and 38.5 % had metastatic disease. Twenty-nine (30 %) patients showed tumor progression, and 20 (21 %) patients died during follow-up. Median progression-free survival (PFS) and overall survival (OS) were 8.6 ± 1.7 and 35.5 ± 1.5 months, respectively. The baseline serum EpCAM levels of the patients were significantly higher than those of the controls (p < 0.001). There was no significant difference in the serum levels of VCAM-1 between the patients and controls (p = 0.47). No significant correlation was detected between the levels of the serum markers and other clinical parameters (p > 0.05). Patients with HER-2-positive and triple-negative tumors had significantly poorer PFS (p = 0.04 and p = 0.001, respectively), while metastatic disease and chemotherapy unresponsiveness had significantly adverse effect on OS analysis (p < 0.001 and p < 0.001, respectively). Neither serum VCAM-1 levels nor serum EpCAM levels were identified to have a prognostic role on either PFS or OS (VCAM-1 p = 0.76 and p = 0.32; EpCAM p = 0.16 and p = 0.69, respectively). Even though any predictive or prognostic role could not be determined for both markers, serum levels of EpCAM were found to have diagnostic value in BC patients.

Is there any diagnostic value of serum protease-activated receptor-1 (PAR1) levels on determination of epithelial ovarian carcinoma?

The role of molecular markers in ovarian cancer is still a matter of debate. Protease-activated receptor-1 (PAR1) might be a good marker in some types of malignant tumors and might provide useful information in diagnosis and prognosis. The objective of this study was to evaluate the serum levels of PAR1 in regard to diagnostic, predictive, and prognostic value in epithelial ovarian cancer (EOC) patients. Forty-four EOC patients were enrolled in this study. Serum PAR1 levels were determined by enzyme-linked immunosorbent assay (ELISA) method. Twenty-five age- and sex-matched healthy controls were included in the analysis. The median age of patients was 58 years old, ranging from 22 to 83 years, where most of them had advanced disease (stage III-IV) (n = 40, 91%). The median serum PAR1 values were significantly elevated in patients compared to healthy controls (1.52 ng/ml vs. 1.13 ng/ml) (p = 0.03), whereas any clinical variables including response to chemotherapy did not associate with serum assay (p > 0.05). Progression-free survival (PFS) and overall survival (OS) of patients who did not respond to chemotherapy nor had platinum resistance in relapsed disease were poorer in the analyses. On the other hand, serum PAR1 levels showed no significant adverse effect on either PFS or OS (p = 0.43 and p = 0.49, respectively). These results proved that baseline serum PAR1 levels of patients with EOC were significantly higher than those of healthy people. However, these assays suggested no predictive or prognostic value in this group of patients.

Clinical significance of serum hepatocyte growth factor (HGF) levels in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the commonest primary malignant cancer of the liver in the world. This study was conducted to investigate the serum levels of hepatocyte growth factor (HGF)in HCC patients and the relationship with tumor progression and known prognostic parameters. Fifty-four patients with HCC were investigated. Pretreatment HGF levels were employed the quantitative sandwich enzyme immunoassay technique (ELISA). Age and sex matched 20 healthy controls were included in the analysis. The median age of the patients was 60 years (range 36-77 years); where males consistituted of majority of the group (88.8%). All of patients had cirrhotic history. Fourty-six percent (n = 25) of patients had Child-Pugh Score A, 30% (n = 16) had Score B or C. All of the patients were treated with local therapies but none of them received sorafenib. The baseline serum HGF levels were significantly higher in patients with HCC than in the control group (p < 0.001). Male patients had higher serum HGF levels compared with female patients (p = 0.01). Serum HGF levels were significantly higher in the patients with elevated serum ALT levels than others with normal serum ALT levels (p = 0.05). Poor performance status (p < 0.001), viral etiology of cirrhosis (p = 0.03), larger tumor size (p = 0.01), lower serum hemogloblin levels (p = 0.03), and not be treated for HCC (p = 0.001) related to worse survival. However, serum HGF did not have significantly adverse effect on survival (p = 0.58). Despite serum HGF levels were found diagnostic value, serum HGF levels had no prognostic value in patients with HCC.

Clinical significance of serum circulating insulin-like growth factor-1 (IGF-1) mRNA in hepatocellular carcinoma.

The principal aim of our study was to investigate the usefulness of serum protein and circulating mRNA of insulin-like growth factor-1 (IGF-1) as a diagnostic and prognostic tool in hepatocellular carcinoma (HCC). Fifty-four HCC patients and age- and sex-matched 20 healthy controls were enrolled into this study. Pretreatment serum IGF-1 and IGF-1 mRNA were determined by the solid-phase sandwich ELISA and quantitative RT-PCR method, respectively. The median age at diagnosis was 60 years, range 36-77 years; where majority of group were male (n = 48, 88.8%). All patients had cirrhotic history. Forty-six percent (n = 25) of patients had Child-Pugh score A, 30% (n = 16) had score B or C. All of the patients were treated with local therapies and none of them received sorafenib. The baseline serum IGF-1 mRNA levels were significantly higher in HCC patients than in the control group (p = 0.04), whereas no significant difference was observed for IGF-1 protein levels between the two group (p = 0.18). Patients with history of HBV infection, who were not treated, and who received multiple palliative treatment for HCC had higher serum IGF-1 mRNA levels (p = 0.03, 0.03, and 0.05, respectively). Poor performance status (p < 0.001), viral etiology of cirrhosis (p = 0.03), larger tumor size (p = 0.01), lower serum hemoglobin levels (p = 0.03), and not be treated for HCC (p = 0.001) related to worse survival. However, neither serum IGF-1 nor serum IGF-1 mRNA had significantly adverse effect on survival (p = 0.53 and 0.42, respectively).

The role of surgical methods in the treatment of anorectal malignant melanoma (AMM).

PURPOSE: Anorectal malignant melanoma (AMM) is a rare tumor with a poor prognosis. The aim of this study was to investigate the clinicopathological characteristics and treatment outcomes in patients with AMM. METHODS: The study included 21 patients diagnosed with AMM between 2000 and 2010 that were evaluated with regard to age, sex, disease stage, treatment modality, and survival. Stage I, II, and III were defined as localized primary malignant melanoma, regional lymph node metastasis, and distant metastasis, respectively. RESULTS: In all, 12 (57%) patients were female and 9 (43%) were male ; median age was 61 years (range : 30-84 years). Among the 21 patients, 7 (47%) underwent abdominoperineal resection and 8 (53%) were treated using wide local excision. Four (19%) patients were classified as stage I, 10 (48%) as stage II, and 7 (33%) patients as stage III. In total, 10 patients received adjuvant therapy. Median overall and progression-free survival was 12 and 9 months, respectively. The 1-year and 5-year overall survival estimates were 59% and 42%, and progression free survival were 49% and 7%, respectively. Patients aged > 60 years (P = 0.145), female patients (P = 0.076), patients with localized disease (P = 0.045), patients that underwent wide local excision (P = 0.619), and patients that received adjuvant therapy (P = 0.962) had longer survival. CONCLUSIONS: The prognosis of AMM remains very poor and disease stage is the only predictor of survival. Abdominoperineal resection does not confer an advantage, in terms of survival, in patients with AMM.

Transdermal methyl alcohol intoxication cause of pain relief.

A 60-year old female patient was found comatosed at home and taken to the hospital's Emergency Department by her relatives. It was learnt that she wrapped her knees with spirit-impregnated cotton pad for pain for one week. On physical examination, only a colour change of purple violet on her knees was noted. Metabolic acidosis with increased anion gap was detected by arterial blood analysis. The patient underwent haemodialysis. She was discharged from the hospital with no complaints, alert and rational following five days of follow-up treatment, with the diagnosis of methyl alcohol poisoning.

Evaluation of flow and structure abnormalities of splanchnic system veins in cirrhotic patients without portal hypertension.

AIM: The aim of this study was to evaluate hemodynamic and anatomic alterations of the splanchnic venous system and the efficiency and safety of color Doppler radial endosonography in the assessment of cirrhotic patients by comparing Child A cirrhotic patients without portal hypertension findings versus a non-cirrhotic group. METHODS: The study was carried out between January 2009 and February 2010; the study population was 38 cirrhotic patients without portal hypertension and 140 control patients. RESULTS: Hepatopedal flow was monophasic in all the control patients; the flow pattern was chaotic and irregular in 8% of the cirrhotic patients; in the cirrhotic patients the portal vein diameter was increased and the flow velocity reduced; no differences in flow volume were observed between the two groups. Splenic vein diameter and flow velocity were normal. In the majority of the Child A cirrhotic patients without portal hypertension, no changes were seen in portal vein diameter or flow volume; in some patients no significant increase portal vein diameter was observed and showed the flow volumes were unchanged. CONCLUSION: Radial Doppler endosonography may be efficient and safe for assessing the splanchnic system.

Potential role of matrix metalloproteinase-2,-9 and tissue inhibitors of metalloproteinase-1,-2 in exudative pleural effusions.

PURPOSE: To investigate diagnostic values of pleural fluid matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitors of metalloproteinase-1 (TIMP-1) and TIMP-2 measurements in tuberculous pleurisy(TP) and malignat pleurisy (MP). METHODS: The study included 24 patients with TP, 22 patients with MP and 15 patients with pleural effusion of non-tuberculous and non-malignant origin as controls. MMP-2,-9 and TIMP-1,-2 levels in pleural fluid were measured by ELISA method. RESULTS: Pleural fluid MMP-2 and MMP-9 levels were higher (P < 0.001, P < 0.001, respectively) in TP than in MP and controls. MP patients have higher pleural fluid MMP-2 and MMP-9 levels (P < 0.01, P < 0.05, respectively) than controls. Pleural fluid TIMP-2 levels were higher (P < 0.01 and P < 0.001, respectively) in MP than in TP and controls. Pleural fluid MMP-9 levels were negatively correlated with pleural fluid TIMP-2 levels (r: 0.464, P=0.029) in patients with MP. CONCLUSIONS: Determination of TIMP-2 in pleural fluid may contribute to differentiate TP from MP. These results suggest that overproduction of MMP-9 and TIMP-2 is associated with accumulation of the pleural effusion in malignancy. Further studies with a greater number of patients are needed to confirm this hypothesis.

Therapeutic and protective effects of autologous serum in amikacin-induced ototoxicity.

OBJECTIVE: Possible therapeutic and protective benefits of intratympanic autologous serum application in amikacin-induced ototoxicity were investigated. METHODS: Twenty-four guinea pigs were separated equally into two groups: therapeutic (group A) and protective (group B). Transient evoked otoacoustic emissions were recorded before and after autologous serum application. Apoptotic cells were identified in the organ of Corti, spiral limbus and spiral ganglion by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling ('TUNEL') method. RESULTS: Transient evoked otoacoustic emission responses at 1, 1.4 and 2.8 kHz improved without significance after autologous serum application in group A (p > 0.05). A significantly protective effect of autologous serum was determined at 4 kHz in group B (p < 0.05). There were significantly fewer apoptotic cells at the spiral limbus in the therapeutic and protective groups compared to the control group (p < 0.05). CONCLUSION: Autologous serum may offer protection against ototoxicity-induced hearing loss, but it cannot restore hearing. Immunohistochemically, autologous serum significantly decreases activation of the intrinsic pathway of pro-apoptotic signalling in mesenchymal cells compared to neurons and neurosensory cells.

Effect of recombinant human activated protein C on apoptosis-related proteins.

The recombinant human activated protein C (rhAPC) has been reported to reduce mortality in patients with severe sepsis. An anti-apoptotic effect of rhAPC in sepsis is known, but the mechanism through which it acts on the apoptotic pathway is still unclear. Therefore, immunopositivity of the apoptosis-related proteins Bcl-2, an anti-apoptotic protein, c-myc, a proliferative protein, p-21 and p-53, two apoptotic proteins, was determined after rhAPC treatment in a mouse sepsis model. Sepsis was induced by Escherichia coli endotoxin injection. Increased neutrophil infiltration and immunoreactivity to p53 and p21 were observed in the group with sepsis and these immunoreactivities were decreased by rhAPC treatment. In the sepstic group; immunopositivity of Bcl-2 and c-myc was mild and moderate, respectively. In conclusion; p21- and p53-mediated apoptosis was increased in the sepsis model, and for the first time it has been shown that rhAPC decreases sepsis-induced apoptosis resulting from increased p21 and p53 proteins.

Anti-cancer efficiency of natural killer cells differentiated from human adipose tissue-derived mesenchymal stem cells and transfected with miRNA150.

AIM: The aim of this study is to investigate the effects of miR150 transfection on NK-like cells differentiated from adipose tissue derived mesenchymal stem cells (AD-MSCs). METHODS: NK-like cells were differentiated from AD-MSCs and activated by miR150 transfection. Transfected/non-transfected NK-like cells were characterized by immunohistochemical and RT-PCR analyzes. Apoptotic efficiency of the transfected/non-transfected NK-like cells on pancreatic cancer cells PANC1 were determined by TUNEL and RT-PCR. RESULTS: In miR150-transfected cells, the increased expression of NK cell-specific genes such as GKMB, KIR2DL2, CD16, CD56, NKG2D, NKp46 and increased immunoreactivity of NK cell-specific surface marker CD314 (NKG2D) were evident. TUNEL assays showed that NK-like cells with/without transfection induced apoptosis in PANC1 cells in the same manner. The decrease in oncogene expression and the increase in the tumor suppressor gene expression in PANC1 cells upon co-culture with NK-like cells differentiated from AD-MSCs were more prominent following miRNA150 transfection. CONCLUSION: It was shown in vitro that NK-like cells could be obtained by differentiation from AD-MSCs and their efficiency could be increased via miR150 transfection. The results are encouraging for further clinical studies in improvement of immunotherapeutic approaches for cancer therapy.

Chemotherapy influences inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) activity on 3D breast cancer cell line.

Multicellular tumor spheroids (MTS) are three-dimensional structural forms of tumors grown in vitro in the laboratory. In this study, the aim was to determine the regulation of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) expressions on MTS in response to treatment with the commonly used anti-cancer drugs Doxorubicin and Docetaxel. The spheroids were generated using the "liquid overlay" technique. The distribution of both iNOS and eNOS was detected using indirect immunohistochemistry, while the expression of both iNOS and eNOS was measured using Western blots. Additionally, S-phase analysis using 5-bromo-2'-deoxyuridine (BrdU) was done on the MTS after treatment with doxorubicin, docetaxel, and a combination of the two. The Griess method was used to measure nitric oxide (NO) production in the cells. An increase in iNOS immunoreactivity and a decrease in eNOS immunoreactivity were observed after doxorubicin treatment, when compared with the other groups. Furthermore, upregulation of iNOS and downregulation of eNOS were detected in doxorubicin-treated cells using Western blotting. Insignificant iNOS expression was observed in all of the groups, and it was particularly low in the control and drug combination groups. NO production was also found to be significantly high after docetaxel treatment, and cell proliferation decreased after doxorubicin treatment. In conclusion, chemotherapy influences NOS activity differently with the presence of different drugs. The results with iNOS show that doxorubicin is a more effective drug than docetaxel, and a drug combination may play a helpful role in the suppression of tumorigenicity and cancer metastasis. Interestingly, eNOS expression increased after the addition of both docetaxel and the drug combination, and it was found to negatively correlate with the histological grade of the tumor. Therefore, analyzing the expression of both iNOS and eNOS might be very useful for targeting the treatment of breast carcinoma and obtaining better information on prognosis.

Effect of apoptosis and response of extracellular matrix proteins after chemotherapy application on human breast cancer cell spheroids.

Multicellular tumor spheroid (MTS) represents a three-dimensional structural form of tumors in laboratory conditions, and it has the characteristics of avascular micrometastases or intervascular spaces of big tumors. Recent studies indicate that extracellular matrix (ECM) proteins play a critical role in tumor metastasis, therefore normal and cancer cells require an ECM for survival, proliferation and differentiation. Doxorubicin and Docetaxel are widely used in the therapy of breast cancer, as well as in in vivo and in vitro studies. In this study, we examined the effect of apoptosis and proliferation of cells on the human breast cancer cell line, MCF-7, by using p53, bcl-2 and Ki67 gene expression, and the tendency to metastasis with extracellular matrix proteins, laminin and type IV collagen after chemotherapy in the spheroid model. The apoptotic cell death in situ was detected by TUNEL method. TUNEL-positive cells and positive immunoreactivities of laminin, type IV collagen, p53 and, bcl-2 were detected in the control group. There was no laminin and type IV collagen immunoreactivities in spheroids of drug groups. While TUNEL-positive cells and p53 immunoreactivity were detected in Docetaxel, Doxorubicin and Docetaxel/Doxorubicin groups, p53 immunoreactivity was not observed in the Docetaxel group. There was no bcl-2 immunoreactivity in either drug group. In addition, we did not detect Ki67 immunoreactivity in both control and drug treatment groups. However, the absence of Ki67 protein in MCF-7 breast multicellular tumor spheroids is possibly related to the cells in G0 or S phase. These chemotherapeutic agents may affect the presence of ECM proteins in this in vitro model of micrometastasis of spheroids. These findings suggest that the possible mechanism of cell death in Doxorubicin and Docetaxel/Doxorubicin treatment groups is related to apoptosis through the p53 pathway. However, we considered the possibility that there is another control mechanism for the Docetaxel group.