Search

Introduction Integrin signalling regulates proliferation, migration, metastasis and cell death and has been suggested as a possible target in antitumor therapy. The aim of this study was to investigate the potential of different integrin α subunits knockdown in increasing sensitivity of triple-negative breast cancer (TNBC) and melanoma cell lines to cisplatin, microtubule poisons paclitaxel (PTX) and vincristine (VCR), and mitigating metastatic potential. Material and methods The knockdown of integrin subunits αv, β3, β5, α3 and α4 was achieved by transfection of siRNA. The sensitivity of cell lines to anticancer drugs was determined using MTT assay. For monitoring cell migration and invasion, migration or matrigel-coated invasion Transwell Cell Culture Inserts were used. Flow cytometry, western blot, and confocal microscopy were done using standard protocols. Results and discussions We analysed the proof of concept in melanoma cell line MDA-MB-435S that expresses integrins αvβ3 and αvβ5 but not αvβ1. Following transfection with integrin- specific siRNAs integrin subunit αv knockdown was selected since it increased sensitivity to PTX and VCR, and decreased metastatic potential. We observed an integrin switching effect upon β3, β5 and α4 knockdown that led to differing changes of sensitivity to antitumor drugs and cell migration. Using a panel of TNBC (MDA-MB-231, –468 and −436) and melanoma (RPMI- 7951, MeWo and A375) cell lines we showed that knockdown of integrin αv increased sensitivity to PTX and VCR and inhibited metastatic potential in MDA-MB-231, MDA-MB-468, RPMI-7951 and MeWo, while no effect was observed in MDA- MB-436 and A375 cells. To assess the importance of integrins αvβ3/β5 in sensitisation to PTX we exposed all cell lines to combination of αvβ3/ β5 inhibitor cilengitide and PTX and observed increased sensitivity to PTX in MDA-MB- 231, MDA-MB-436, RPMI-7951 and A375 but surprisingly decreased sensitivity in MeWo and MDA-MB-468 cell lines, implying either involvement of other integrins αv or integrin switching effect. The correlation between loss of phosphorylated focal adhesion kinase pFAK(Y397) and increased sensitivity to PTX was observed in melanoma cell lines, however, sensitisation was not achieved using pFAK(Y397) inhibitor. Conclusion Our results highlight that knocking down integrin αv, rather than inhibiting αvβ3/ β5 using cilengitide, could be more effective for increasing sensitivity to microtubule poisons and decreasing metastatic potential in TNBC and melanoma.

(A) Normalized melt curves in duplicates showing the melt behavior of methylation standards (red=0 %, pink=25 %, blue=50 %, green=75 %, orange=100 %) and an unknown sample (black). (B) Regression model used for MGMT promoter methylation quantification. Area under the curve (AUC) from the normalized melt curves are used and regressed to the known methylation level of the standards. The linear regression model was chosen for quantification (R² > 0.98). (C) Pyrogram of the MGMT promoter of a patient tumor sample with a mean methylation of 31.4 %. (D) Typical pyrogram obtained from a grade III patient tumor sample indicating a heterozygous G-to-A point mutation of the IDH1 gene resulting in a mutation at codon 132 (R132H). (E) Relationship between MGMT protein activity and promoter methylation (r3) in 14 GBM cell lines. Figure S2. (A) Pyrogram of the whole HRM (R4) assay region from a patient sample. (B) Dot-plot of the methylation values obtained in a subsample by HRM and pyrosequencing showing a high correlation of this two methods. The dotted line indicates the 95 % CI. Figure S3. Overlay of Kaplan-Meier estimates of PFS and OS according to MGMT promoter methylation status. Kaplan-Meier estimates for (A) PFS and (B) OS of 65 high-grade glioma patients determined by HRM (red lines), MSP (blue) and PSQ (green). The solid and the dashed lines indicate the group as being categorized unmethylated (UM) or methylated (ME), respectively. Table S1. Primers used for HRM and pyrosequencing. Table S2. MGMT promoter methylation status determined by HRM, MSP, and PSQ in dependence of the IDH1 status. Table S3. ROC curves were plotted for 15 methylation cut-off scores (1-15 %) for predicting PFS ≥ 12 months and OS ≥ 18 months. (PPTX 312 kb)

Triple-negative breast cancer (TNBC) do not express estrogen receptor, progesterone receptor or human epidermal growth factor receptor 2 (Her2) and therefore, chemotherapy remains the mainstay of treatment. Integrin signalling regulates numerous processes in tumor cells including cell adhesion, migration, invasion and survival. Integrins are used as prognostic indicators in breast cancer. The aim of this study was to investigate whether knockdown of integrin αv could increase sensitivity to vincristine, paclitaxel and cisplatin, and inhibit cell migration, which is a prerequisite for cell invasion. Expression of integrin subunit αv and integrin heterodimers αvβ3 and αvβ5 in metastatic MDA-MB-435S MDA-MB-231, MDA-MB-468 and MDA-MB-436 cells was measured by flow cytometry. All cells expressed αv integrin subunit. Integrin heterodimer αvβ5 was highly expressed in all cells except MDA-MB-436. Integrin heterodimer αvβ3 was highly expressed in MDA-MB-435S and MDA-MB-231, while a lower amount was found in MDA-MB-468 while MDA-MB-436 cells were found to be αvβ3 negative. Decreased expression of integrin αv, achieved by αv-specific siRNA transfection, increased sensitivity of MDA-MB-435S, MDA-MB-231 and MDA-MB-468 cells to vincristine and increased sensitivity of MDA-MB-435S and MDA-MB-231 cells to paclitaxel. Conversely, decreased expression of integrin αv decreased sensitivity to cisplatin in MDA-MB-435S and MDA-MB-468 cells but increased sensitivity in MDA-MB-468 cells. Upon integrin αv knockdown in MDA-MB-436 cell line no alteration in sensitivity to any of the abovementioned drugs was observed. The in vitro transwell migration assay in MDA-MB-435S, MDA-MB-231 and MDA-MB-468 cells showed that knockdown of integrin αv strongly decreases cell migration. The most dramatic effect was observed in MDA-MB-435S and MDA-MB-468 cells. Immunofluorescence analysing in MDA-MB-435s cells showed disappearance of actin stress fibers and focal adhesion marker protein phospho-paxillin (Y113). We suggest that transfection of at least some TNBC cells with integrin αv siRNA could enhance activity of vincristine and paclitaxel, and simultaneously inhibit their metastatic potential. These new insights should be useful when devising strategies for more efficient TNBC treatment.

Objective: MGMT promoter status is an important prognostic factor in high-grade glioma patients and indispensable for decision making in neurooncological therapy. Currently, the MGMT promoter methylation is determined by methylation-specific PCR (MSP). The development of new approaches to improving [for full text, please go to the a.m. URL], 66. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC)

Integrin signalling regulates numerous cellular processes in tumor cells including tumor cell adhesion, migration, invasion and survival. Integrins play an important role in breast cancer metastasis. In addition, metastatic cancer cells are highly resistant to antitumor drugs. The aim of this study was to investigate the role of integrins αvβ3, αvβ5, α3β1 and α4β1 expressed on MDA-MB-435S cells in sensitivity to different antitumor drugs and cell migration, one of the necessary requirements for efficient cell invasion and metastasis. For silencing of integrin subunits β3, β5, αv, α3 and α4 in human breast carcinoma MDA-MB-435S cell line, the siRNA sequences specific for β3, β5, αv, α3 and α4 were transfected using Lipofectamine RNAiMAX Reagent. Flow cytometry analysis was used to analyse the expression of integrins αvβ3, αvβ5, αv, α3β1 and α4. Confocal microscopy was used to visualise paxillin, phospho-paxillin or F-actin fibers using specific antibodies or phalloidin-FITC, respectively. Cell survival was assessed using MTT assay. Boyden chambers (pore size 8 μm) were used to measure migration. While decreased expression of integrins αvβ3, αvβ5 or α3β1, achieved by β3, β5, αv or α3-specific siRNA transfection, reduced sensitivity of MDA-MB-435S cells to cisplatin, no change was observed in MDA-MB-435S cells upon knockdown of α4β1 using α4 siRNA transfection. Conversely, decreased expression of integrins αvβ5 or α4β1 upon integrin subunit β5, αv or α4 siRNA transfection sensitized MDA-MB-435S cells to vincristine or paclitaxel, but there was no change in cell survival upon knockdown of integrins α3β1 or αvβ3 upon α3 or β3 siRNA transfection. The cytotoxic activity of camptothecin and doxorubicin was not altered upon transfection of any of analysed siRNAs. The down-regulation of integrins αvβ3 and αvβ5 achieved by αv siRNA transfection strongly decreased migration, amount of stress fibers and focal adhesions, whereas the down-regulation of integrin α4β1 upon α4 siRNA transfection moderately increased cell migration with simultaneous slight increase in amount of stress fibers and focal adhesions. We suggest that transfection of at least some breast carcinoma cells with integrin αv siRNA could enhance activity of vincristine or paclitaxel and simultaneously inhibit their metastatic potential. These new insights should be useful when devising strategies for more efficient treatment.

Integrin signalling regulates numerous cellular processes including proliferation, migration, metastasis, cancer-induced angiogenesis and cell death. Previously it was shown that the patients having MGMT negative tumors can benefit from the cilengitide therapy. This is due to the fact that temozolomide (TMZ) can achieve a maximal cytotoxic effect in those tumors, since TMZ-induced primary DNA damage (O6-Methylguanine) cannot be repaired. The aim of this study was to investigate the role of integrins αvβ3, αvβ5, α3β1 and α4β1 in a putative resistance to TMZ, a monoalkylating agent used in the first-line therapy of malignant gliomas. For this we determined the integrin status (together with MGMT and p53 status) in a set of human adult malignant glioma cell lines, from which four MGMT negative cell lines (LN229, LN308, U87MG and U138MG) were chosen for further experiments. Decreased expression of all examines integrins αvβ3, αvβ5, α3β1 and α4β1 achieved by β3, β5, αv, α3 or α4-specific siRNA transfection sensitized the high-grade glioma cell lines to TMZ irrespective of the p53 status, as determined by MTT, colony formation and sub-G1 (PI) flow cytometric assay. Also, treatment of the cell lines with a cilengitide-similarRGD integrin inhibitir sensitized them to TMZ. Silencing of integrins αvβ3, αvβ5 and α4β1 expressed on LN229 and of αvβ3, αvβ5, α4β1 and α3β1 expressed on U138MG cells led to abrogation of the FAK/ILK/Akt pathway, leading to more pronounced induction of TMZ-induced γH2AX foci (DNA double-strand breaks), reduction in Ead51 protein level and apoptosis, as determined by caspase-3 cleavage and decreased Bcl-xL and survivin expression, in comparison to TMZ alone. Also, subcutaneous U87MG xenografts in mice treated i.p. with the integrin inhibitor and TMZ achieved stronger tumor regression compared to those treated with TMZ or with the integrin antagonist alone. Protein extracts isolated from the ablated tumor xenografts that have been exposed to TMZ and integrin antagonist showed the strongest level of H2AX phosphorilation and caspase-3 cleavage, pointing to increased DNA damage and cell death, again suggesting that integrin silencing impairs DNA repair.

Nalazi se u knjizi sažetaka

Nalazi se u knjizi sazetaka