Downregulation of Rac1/PAK1/LIMK1/cofilin signaling pathway in colon cancer SW620 cells treated with Chlorin e6 photodynamic therapy

BackgroundColorectal cancer is one of the most common gastrointestinal malignancies. Photodynamic therapy (PDT) is a novel and non-invasive treatment for tumors as PDT features small trauma, good applicability, andaccurate targeting. PDT may also be a potential treatment for colon cancer as itmay may induce suppressive effects on metastatic potential.. However, the molecular mechanism of the Chlorin e6 Photodynamic therapy (Ce6-PDT) inhibiting the migration of human colon cancer SW620 cells remains unclear.MethodsScratch wound healing assay, scanning electron microscope, MTT, immunofluorescence and laser confocal technique were used to investigate the suppressive effects of Ce6-PDT on the SW620 cells migration, pseudopodia, viability and the actin cytoskeleton. The effect of Ce6-PDT on actin-Filaments and signaling molecules of the Rac1/PAK1/LIMK1/cofilin signaling pathway in SW620 cells were examined by western blot analysis. RNA interference (RNAi) technology was used to establish siRNA-Rac1/SW620 cells. The combined effects of Ce6-PDT and RNAi on colon cancer SW620 cells was investigated by the same technology and methods mentioned above to clarify the signal transduction effect of Rac1/PAK1/LIMK1/cofilin signaling pathway in Ce6-PDT caused inhibition of SW620 cell migration.ResultsThe healing and migration rate of the SW620 cells was significantly reduced and the cell pseudopodia were reduced or disappeared by Ce6-PDT. The Immunofluorescence and western blot analysis results showed that Ce6-PDT destroy microfilament’s original structure and significantly downregulated F-actin protein expression. The Rac1/PAK1/LIMK1/cofilin signaling pathway was downregulated by Ce6-PDT. Furthermore, the RNAi significantly strengthened the effect of Ce6-PDT on colon cancer SW620 cells migration.ConclusionsActin cytoskeleton and protrusions of SW620 cells correlate with its migration ability. Ce6-PDT suppresses SW620 cells migration by downregulating the Rac1/PAK1/LIMK1/cofilin signaling pathway, and its suppressive effect was enhanced by knocking down Rac1 gene expression.     

Up-regulation of ABCG2 by MYBL2 deletion drives Chlorin e6-mediated photodynamic therapy resistance in colorectal cancer

ObjectivePhotodynamic therapy (PDT) may be an effective therapeutic strategy for colorectal cancer at an early stage. However, malignant cells’ resistance to photodynamic agents can lead to treatment failure. MYBL2 (B-Myb) is an oncogene in colorectal carcinogenesis and development, for which little research has focused on its effect on drug resistance.Materials and MethodsIn the present work, a colorectal cancer cell line with a stable knockdown of MYBL2 (ShB-Myb) was constructed first. Chlorin e6 (Ce6) was utilized to induced PDT. The anti-cancer efficacy was measured by CCK-8, PI staining, and Western blots. The drug uptake of Ce6 was assayed by flow cytometry and confocal microscopy. The ROS generation was detected by the CellROX probe. DDSB and DNA damage were assayed through comet experiment and Western blots. The over-expression of MYBL2 was conducted by MYBL2 plasmid.ResultsThe findings indicated that the viability of ShB-Myb treated with Ce6-PDT was not decreased compared to control SW480 cells (ShNC), which were resistant to PDT. Further investigation revealed reduced photosensitizer enrichment and mitigated oxidative DNA damage in colorectal cancer cells with depressed MYBL2. It turned out that SW480 cells knocking down MYBL2 showed phosphorylation of NF-κB and led to up-regulation of ABCG2 expression thereupon. When MYBL2 was replenished back in MYBL2-deficient colorectal cancer cells, phosphorylation of NF-κB was blocked and ABCG2 expression up-regulation was suppressed. Additionally, replenishment of MYBL2 also increased the enrichment of Ce6 and the efficacy of PDT.ConclusionIn summary, MYBL2 absence in colorectal cancer contributes to drug resistance by activating NF-κB to up-regulate ABCG2 and thereby leading to photosensitizer Ce6 efflux. This study provides a novel theoretical basis and strategy for how to effectively improve the anti-tumor efficacy of PDT.     

Chlorin e6 mediated photodynamic therapy triggers resistance through ATM-related DNA damage response in lung cancer cells

ObjectivePhotodynamic therapy (PDT) is a promising strategy for the treatment of malignant tumors due to its high selectivity, limited-toxicity, and non-invasiveness. However, PDT can also induce DNA damage and subsequent repair response, which may reduce the efficacy of PDT. In the present study, we sought to explore the effect of chlorin e6 (Ce6)-mediated PDT on DNA damage and DNA damage response (DDR) in lung cancer cells. In addition, the effect of PDT combined with ATM inhibitor on molecules of DDR and the possibility of improving the efficacy of PDT were further investigated.Materials and methodsIn the in vitro study, lewis cells were submitted to Ce6 treatment (2, 4, 8, 16, 32 μg/mL). To determine the concentration of Ce6, uptake and toxicity of Ce6 mediated PDT were detected using flow cytometry (FACS), Confocal microscopy, and CCK-8. In the subsequent research, 8 μg/mL of Ce6 was the treatment condition for inducing PDT. The different post-irradiation placement times were further grouped under this condition (2, 4, 6, 12 h). Cellular reactive oxygen species (ROS), damage of DNA were measured by DCFH-DA probe, comet assay respectively. Then the expression of p-ATM, p53, and γ-H2A.X proteins related to DNA damage response, was detected by WB. The efficacy of Ce6 induced PDT was also demonstrated by Annexin-V/PI staining as well as the expression of PCNA, cleaved-caspase-3. On this basis, ATM inhibitor was applied to treat lewis cells combined with Ce6 (2, 4 h) to investigate whether the efficacy of PDT induced by Ce6 can be improved after the ATM-related DDR was blocked. The cell viability, apoptosis, and expression of associated proteins were assayed.ResultsAt 2–4 h after PDT treatment, ROS was dramatically elevated in lewis cells, DNA double-strand breaks (DDSB) occurred, as well as up-regulation of DDR proteins γ-H2A.X, p-ATM, and p53. At the same time, lewis cells did not undergo significant apoptosis. After ATM inhibition, the DDR was significantly blocked within 2–4 h after Ce6 induced PDT, along with a pronounced decrease in cell viability followed by a prominent increase of apoptosis.ConclusionCe6-mediated PDT generates ROS in a short period time, thus inducing DNA damage, ATM-related DDR as well as promoting resistance of lung cancer cells to PDT. Combining ATM inhibitor with PDT could effectively inhibit the DDR induced by PDT, thereby enhancing the efficacy. This study reveals a new resistance mechanism of PDT and proposes an intervention strategy.     

The role of autophagy in the treatment of colon cancer by chlorin e6 photodynamic therapy combined with oxaliplatin

BackgroundPhotodynamic therapy is a tumour treatment method. Its mechanism mainly induces apoptosis, autophagy, and other ways to cause cell death. Therefore, this study aims to evaluate the therapeutic effect of chlorine e6 photodynamic therapy (Ce6-PDT) combined with oxaliplatin (L-OHP) in colon cancer and to investigate the role of autophagy in L-OHP treatment and Ce6-PDT combined with L-OHP in colon cancer.MethodsCCK-8 assay, Scratch wound healing assay, and Western Blot (WB) were used to identify drug-resistant colon cancer cell line SW620/L-OHP. Annexin V/FITC assay, laser confocal double immunofluorescence staining method and WB were employed to investigate the apoptosis and autophagy changes in Ce6-PDT combined with L-OHP.ResultsDrug resistance cells SW620/L-OHP were developed under the continuous multi-generation of L-OHP treatment, and the expression of ATP-binding cassette subfamily B member 1 (ABCB1) and ATG5 proteins were increased. The results of immunofluorescence showed that LC3B accumulated in SW620 cells and SW620/L-OHP cells under the treatment of L-OHP. The WB results indicated that LC3B and ATG5 protein expression was increasing in SW620 cells and SW620/L-OHP cells. Inhibition of L-OHP-induced autophagy reduces SW620 cells and SW620/L-OHP cells’ viability while increasing apoptosis and the Pro Caspase-3 protein expression. The combination of Ce6-PDT and L-OHP decreased the cell viability, the cell migration ability, the Bcl-2 protein expression, and increased the apoptosis rate, Pro Caspase-3 protein expression in SW620 cells.ConclusionsL-OHP can cause SW620 cells drug resistance. Autophagy plays a protective role in the L-OHP treatment of SW620 cells and SW620/L-OHP cells, and inhibition of autophagy can increase the efficacy of L-OHP. Ce6-PDT combined with L-OHP can further improve the tumor's therapeutic effect, and autophagy inhibition can improve the efficacy of combined therapy.     

Novel piperazine-substituted silicon phthalocyanines exert anti-cancer effects against breast cancer cells

BackgroundPhotodynamic therapy (PDT) is one of the effective methods that can be used in cancer treatment. In this study, we aimed to investigate the PDT-mediated anti-cancer effects of newly synthesized piperazine-substituted silicon phthalocyanine molecules on breast cancer cells.MethodsThe compounds were analyzed by different spectroscopic techniques (FT-IR, UV–vis, ¹H NMR, ¹³C NMR, MS) and the absorbance characteristics were determined. The cytotoxic effects of silicon phthalocyanines on MDA-MB-231 breast cancer cells and non-tumorigenic MCF-10A cells were evaluated using MTT assay. Detection of apoptotic populations was performed by Annexin V/7AAD assay. H₂DCFDA dye was used to analyze intracellular reactive oxygen species. The clonogenic activity and cellular motility were analyzed by colony formation assay and in vitro scratch assay, respectively. Caspase-3, PARP1, and cleaved-PARP1 protein levels were analyzed by western blot studies.ResultsPiperazine-substituted silicon phthalocyanines caused high levels of cytotoxic effects and apoptotic cell population in MDA-MB-231 cells, while low levels of cytotoxic effects were observed in MCF-10A cells. Following PDT, intense ROS formation was detected in MDA-MB-231 cells. Colony-forming capacity and cellular motility of MDA-MB-231 cells were highly restricted following PDT, whereas these effects were observed at lower levels in MCF-10A cells. Silicon phthalocyanines caused different effects on cleaved-PARP1 expressions of MDA-MB-231 and MCF-10A cells.ConclusionThese results suggest that piperazine-substituted silicon phthalocyanines can exert selective anti-cancer effects on breast cancer cells and activate cellular death through different molecular pathways. Hence, we believe that they may be used as effective photosensitizer agents in the future.     

Inhibition of cell growth induced by photosensitizer PP(Arg)2-mediated photodynamic therapy in human breast and prostate cell lines. Part I

Photodynamic therapy can become an effective alternative method to surgery. The experiments reveal that using low photosensitizer doses and relatively low energy doses allow us to obtain effective results after PDT (to limit formation of colonies by investigated cancer cells). The prostate and breast cancer cell lines were investigated: MCF-7, a human breast cancer responsive to androgen therapy; MDA-MB231, a more aggressive human breast cancer non-responsive to androgen therapy; LNCaP, a lymphonodal metastasis of prostate carcinoma responsive to androgen therapy; DU-145, a human prostate cancer non-responsive to androgen therapy. Clonogenic assay shows that certain PP(Arg)(2) and light energy low doses stimulate the researched colony-forming cancer cells growth. Some low energy doses used during PP(Arg)(2)-mediated PDT also cause the increase in the colony-forming tumor cells. Among investigated cancer lines, MCF-7 exhibited the biggest sensibility towards PP(Arg)(2) and LNCaP the smallest one. PP(Arg)(2) based PDT is an effective method in colony growth limitation of breast cancer cell lines: MCF-7, MDA-MB231 and prostate cancer cell lines: LNCaP, DU-145.     

Anticancer effects elicited by combination of Rubus extract with phthalocyanine photosensitiser on MCF-7 human breast cancer cells

BackgroundPhotodynamic therapy (PDT) is a novel approach for the treatment of cancer and other related diseases. Breast cancer remains the most common cause of cancer-related death in women. This study was carried out to investigate the photosensitizing capacity of Rubus fairholmianus root acetone extract (RFRA) in vitro.MethodsRFRA was coupled with phthalocyanine photosensitizer to enhance the therapeutic properties on MCF-7 breast cancer cells. Comparatively low dose photosensitizer (PS) and Rubus extract have been used for the conjugation as it induces cell death at low doses. The diode laser of wavelength 680 nm and 5, 10 and 15 J/cm2 fluencies have been used for PDT experiments/laser irradiation. MCF-7 cells were exposed to Rubus extract and conjugated Rubus-PS for 24 h and analysed the alterations in cell morphology, proliferation, cytotoxicity and apoptosis induction.ResultsThe PDT-treated cells displayed substantial features of apoptotic cell death by changes in morphology with a reduction in cell number, development of apoptotic bodies and cell detachment from culture plates. Cellular viability (51.25% for RFRA-PS at 15 J/cm2) and Adenosine 5′-triphosphate (ATP) proliferation of treated cells reduced significantly and the cytotoxicity increased in lactate dehydrogenase (LDH) assay. The Annexin V/PI double staining supports the caspase 3/7 activities by the increased apoptotic cells population and the increased levels of cytochrome c.ConclusionOur results show that the phototoxic properties of RFRA and photosensitizer may be through the caspase-mediated apoptosis and it can be summarised that Rubus may be a potent anticancer plant with phototoxic effects on breast cancer cells.     

三氧化二砷逆转乳腺癌细胞株MCF-7/ADM多药耐药的研究

目的探讨三氧化二砷（As2O3）体外逆转人乳腺癌细胞多药耐药性的作用及机制。方法采用MTT法检测As2O3的细胞毒作用和处理前后耐药细胞对化疗药物的敏感性,用流式细胞仪检测细胞内阿霉素浓度,通过RT-RCR检测MDR1基因的表达。结果 As2O3在0.25mg/L以下剂量时对MCF-7和MCF-7/ADM耐药细胞株的抑制率均小于15%,半数抑制率（IC50）分别为1.01m/L和1.28mg/L,无细胞毒剂量0.2mg/L的As2O3能部分逆转MCF-7/ADM细胞对阿霉素的耐药性。同时无细胞毒剂量0.2mg/L的As2O3能使MCF-7/ADM细胞内阿霉素浓度明显增加,MDR1表达下降。结论 As2O3具有体外部分逆转人乳腺癌细胞多药耐药性的作用,可能与增加细胞内药物积累、下调MDR1表达有关。     

补骨脂素对MCF-7/ADR耐药细胞内ADR及Ca2+浓度的影响

目的 研究补骨脂素对MCF-7/ADR耐药细胞逆转作用及对耐药细胞内Ca2 浓度影响,探讨其可能作用机制.方法 采用MTT法测定补骨脂素对细胞增殖抑制作用,高效液相色谱法检测细胞内ADR的浓度,激光共聚焦显微镜测定细胞内不同时段的Ca2 浓度.结果 补骨脂素在1～20 μmol/L浓度下能不同程度降低ADR对MCF-7/ADR细胞的IC50,并不同程度提高细胞内ADR浓度;1～20 μmol/L补骨脂素在不同作用时段(24、48、96 h)对MCF-7/ADR细胞内Ca2 浓度与作用时间呈负相关.结论 补骨脂素能逆转MCF-7/ADR细胞的MDR,其作用机制与影响耐药细胞内Ca2 浓度,故而增加细胞内ADR浓度有关.     

Investigation of ROS generating capacity of curcumin-loaded liposomes and its in vitro cytotoxicity on MCF-7 cell lines using photodynamic therapy

Photodynamic therapy (PDT) is highly efficient in eradicating targetlesions by using photosensitizers (PS) triggered by external light energy. Nanotechnology may help increase the solubility and effective delivery of PS towards improving its efficacy. Curcumin (Cur) was used as a natural PS for PDT in the present work. Briefly, curcumin was encapsulated in liposomes (LPs) using the thin film hydration method and optimized using the QbD approach through the Box-Behnken Design (BBD) to optimize the responses like entrapment efficiency and drug loading with a smaller vesicle size. The in vitro release studies performed using a dialysis bag (MWCO 12 KDa) suggested a sustained release of the Cur over 72 h in pH 7.4 PBS following the Weibull drug release kinetics. In addition, the ROS generating capabilities upon application of blue light (460 nm) and resulting cytotoxicity were evaluated in MCF-7 cell lines. The Cur-loaded liposome exhibited significant ROS generation and cytotoxicity to the cancer cells than free curcumin. Thus, the Cur-loaded liposomes could be used to treat breast cancer with photodynamic therapy.     

Triple-negative breast cancer treatment in xenograft models by bifunctional nanoprobes combined to photodynamic therapy

Triple-negative breast cancer (TNBC) overexpresses the Epidermal Growth Factor Receptor (EGFR), a characteristic of different types of tumors, linked to worse disease prognosis and risk of recurrence. Conventional treatments are also aggressive and can be morbid.. Therefore, t improvement and development of new methods are notorious. Photodynamic Therapy (PDT) is an effective method for treating different types of cancer by using light radiation to activate a photosensitizing agent (drug) in molecular oxygen presence, promoting cell death., Improving drug uptake in target cells could contribute to PDT efficiency. Accordingly, we developed a bifunctional nanoprobe (BN), used in PDT as a a treatment method in vivo against breast cancer. The BN uses gold nanoparticles with active targeting through the Epidermal Growth Factor (EGF) protein and Chlorine e6 (Ce6) carriers. We evaluated the therapeutic efficacy of in vivo xenograft in 4 groups: Saline, BN, Ce6+PDT, and BN+PDT. As a result, we observed that the BN+PDT group exhibited an excellent effect with greater selectivity to tumor tissue and tissue damage when compared to the Saline, BN, and Ce6+PDT groups. The results indicate a potential impact on breast cancer treatment in vivo.. In conclusion, our data propose that the BN developed heightened PDT efficacy through cellular DNA repair effects and tumor microenvironment.     

Exploring the radiosensitizing potential of magnetotherapy: a pilot study in breast cancer cells

Abstract

Aim: To explore the influence of electromagnetic fields (EMFs) on the cell cycle progression of MDA-MB-231 and MCF-7 breast cancer cell lines and to evaluate the radiosensitizing effect of magnetotherapy during therapeutic co-exposure to EMFs and radiotherapy.

Material and methods: Cells were exposed to EMFs (25, 50 and 100?Hz; 8 and 10?mT). In the co-treatment, cells were first exposed to EMFs (50?Hz/10?mT) for 30?min and then to ionizing radiation (IR) (2?Gy) 4?h later. Cell cycle progression and free radical production were evaluated by flow cytometry, while radiosensitivity was explored by colony formation assay.

Results: Generalized G1-phase arrest was found in both cell lines several hours after EMF exposure. Interestingly, a marked G1-phase delay was observed at 4?h after exposure to 50?Hz/10?mT EMFs. No cell cycle perturbation was observed after repeated exposure to EMFs. IR-derived ROS production was enhanced in EMF-exposed MCF-7 cells at 24?h post-exposure. EMF-exposed cells were more radiosensitive in comparison to sham-exposed cells.

Conclusions: These results highlight the potential benefits of concomitant treatment with magnetotherapy before radiotherapy sessions to enhance the effectiveness of breast cancer therapy. Further studies are warranted to identify the subset(s) of patients who would benefit from this multimodal treatment.     

乙醇降低人乳腺癌MCF-7细胞放射敏感性的研究

目的 研究乙醇对人乳腺癌MCF-7细胞放射敏感性的影响及相关机制.方法 将人乳腺癌MCF-7细胞分为4组,对照组(不做任何处理)、乙醇组(乙醇处理)、单纯照射组(6 GyX射线处理)和联合组(乙醇与X射线联合作用).克隆形成法检测50或100 mmol/L乙醇对MCF-7细胞放射敏感性的影响;流式细胞术检测细胞周期变化;Annexin V-FITC法检测细胞凋亡.结果 50和100 mmol/L乙醇处理MCF-7细胞50 h,对生长无明显影响(t--0.82和1.15,P＞0.05);乙醇预处理2h,可明显增加X射线照射后MCF-7细胞克隆形成的能力(t=4.15和10.28,P＜0.05).相比于单纯照射组,乙醇降低了X射线照射诱导的细胞G2/M期阻滞(t=7.18,P＜0.05),以及sub-G1 峰的比例(t =5.39,P＜0.05).而且,Annexin V-FITC检测结果示,乙醇联合X射线照射的细胞晚期和早期凋亡减少(t=4.86和7.59,P＜0.05).结论 乙醇可以增加MCF-7细胞的辐射抗性,其机制可能与降低辐射诱导的细胞G2/M期阻滞及早、晚期凋亡的发生相关.     

Targeting X box-binding protein-1 (XBP1) enhances the sensitivity of HOS osteosarcoma cells to pyropheophorbide- α methyl ester-mediated photodynamic therapy

Photodynamic therapy (PDT), utilizes a photochemical reaction between photosensitizer and light to cause cancer death by generating reactive oxygen species (ROS). X-box binding protein 1 (XBP1), a downstream product of the IRE1α-XBP1 pathway, regulates diverse target genes, including various proto-oncogenes and its overexpression was closely related to the occurrence and progression of malignant tumors. The present study was performed to explore the role of XBP1 in human osteosarcoma HOS cells treated with pyropheophorbide-α methyl ester (MPPα)-mediated photodynamic therapy (PDT) (MPPα-PDT) and its potential mechanisms. The protein IRE1α and XBP1 increased with a time-dependent manner after MPPα-PDT treated, which indicated that MPPα-PDT induced the activation of the IRE1α-XBP1 pathway in HOS cells. Besides, MPPα-PDT treated alone or combined with XBP1 knockdown could both restrain the cell viability, but the latter one has more notable effect, which indicated that XBP1 knockdown may enhance the cell inhibitory effect by MPPα-PDT. Simultaneously, the apoptotic rate measured by flow cytometry (FCM) was increased surprisedly and the expression of apoptosis proteins was increased when knockdown XBP1 under the MPPα-PDT. In addition, antioxidant-related proteins such as the Catalase and SOD1 protein levels decreased, while the intracellular ROS content increased in HOS cells when knockdown XBP1 under the MPPα-PDT. These results suggested that the mechanism of XBP1 mediating resistance in HOS cells might be related to the expression of antioxidant molecules. In summary, this study found that the IRE1α-XBP1 pathway was activated in HOS cells after MPPα-PDT treated, and furthermore, XBP1 knockdown could decrease HOS cell viability through apoptosis and enhance the anti-tumor effect of MPPα-PDT remarkably in the meantime, which related to the regulation of oxidation-antioxidant system.     

Induction of lethal bystander effects in human breast cancer cell cultures by DNA-incorporated Iodine-125 depends on phenotype

Abstract

Purpose: This study uses a three-dimensional cell culture model to investigate lethal bystander effects in human breast cancer cell cultures (MCF-7, MDA-MB-231) treated with ¹²⁵I-labeled 5-iodo-2 -deoxyuridine (¹²⁵IdU). These breast cancer cell lines respectively form metastatic xenografts in nude mice in an estrogen-dependent and independent manner.

Materials and methods: In the present study, these cells were cultured in loosely-packed three-dimensional architecture in a Cytomatrix? carbon scaffold. Cultures were pulse-labeled for 3 h with ¹²⁵IdU to selectively irradiate a minor fraction of cells, and simultaneously co-pulse-labeled with 0.04 mM 5-ethynyl-2′-deoxyuridine (EdU) to identify the radiolabeled cells using Click-iT^® EdU and flow cytometry. The cultures were then washed and incubated for 48 h. The cells were then harvested, serially diluted, and seeded for colony formation. Aliquots of cells were subjected to flow cytometry to determine the percentage of cells labeled with ¹²⁵IdU/EdU. Additional aliquots were used to determine the mean ¹²⁵I activity per labeled cell. The percentage of labeled cells was about 15% and 10% for MCF-7 and MDA cells, respectively. This created irradiation conditions wherein the cross-dose to unlabeled cells was small relative to the self-dose to labeled cells. The surviving fraction relative to EdU-treated controls was measured.

Results: Survival curves indicated significant lethal bystander effect in MCF-7 cells, however, no significant lethal bystander effect was observed in MDA-MB-231 cells.

Conclusions: These studies demonstrate the capacity of ¹²⁵IdU to induce lethal bystander effects in human breast cancer cells and suggest that the response depends on phenotype.     

5-aminolevulinic acid and sodium ferrous citrate decreased cell viability of gastric cancer cells by enhanced ROS generation through improving COX activity

BackgroundMitochondrial dysfunctions are related to cancer development.. 5-aminolevulinic acid (ALA) is used for photodynamic therapy (PDT). In this PDT, protoporphyrin IX (PpIX), which is converted from ALA, can generate reactive oxygen species (ROS) that kill the cancer cell. ALA is also reported to promote cytochrome c oxidase (COX) activity, which can generate ROS itself. Therefore, this study focused on the effect of ALA during PDT. In addition, in the previous study, sodium ferrous citrate (SFC) is reported to increase COX activity. So, this study also aims to improve the COX activity by the addition of SFC that can promote ROS generation, which has a cytotoxic effect.MethodsIn this study, we used ALA and SFC, then evaluated the effects of the treatment on the human gastric cancer cell line MKN45, including the induction of cell death.ResultsThis study showed that treatment with ALA and SFC increases intracellular heme and heme proteins. Moreover, COX activity was promoted, resulting in the production of intracellular reactive oxygen species (ROS), which eventually reduced the cell viability in human gastric cancer cell line MKN45.ConclusionOur study can detect ROS generation with ALA and SFC. Furthermore, we found this generation of ROS has a cytotoxic effect. Therefore, this phenomenon contributes to the effect of PDT.     

Differential response of normal and transformed mammary epithelial cells to combined treatment of anti-miR-21 and radiation

Purpose: MicroRNA miR-21 has emerged as a therapeutic target in the treatment of breast cancer. This study was designed to compare the responses of breast cancer cells and non-transformed breast epithelial cells to a combined regimen of miR-21 inhibition and radiation.

Materials and methods: The MDA-MB-361 (breast cancer) and MCF-10A (non-transformed mammary epithelial) cell lines were used for the comparison in this in vitro study. The stable knockdown of miR-21 was performed by using lentiviral approach. The response of the cells was monitored 4, 24 and 48?h after the irradiation with 0.25 and 2.5?Gy, using sham-irradiated cells as controls. The response of the cells was established by performing various functional assays – cell viability and cell attachment, clonogenic survival, cell cycle analysis and 3D microtissue formation.

Results: The knockdown of miR-21 induced significant increase in apoptosis and growth delay in MDA-MB-361 cancer cells compared to non-transformed MCF-10A cells. After combined radiation and anti-miR-21 treatment, MDA-MB-361 cells show reduced cell growth and viability what is presented in their inability to form colonies. MCF-10A cells were not as sensitive to the combined treatment and that has also been confirmed with colony forming assay.

Conclusions: Cellular response to a combined treatment of anti-miR-21 and radiation is different between cancer and non-cancer cells which highly support the idea of linking miR-21 inhibitor and radiation treatment in the future therapeutic approaches for breast cancer.     

The evaluation of radio-sensitivity of mung bean proteins aqueous extract on MCF-7, hela and fibroblast cell line

Purpose: Breast cancer is one of the most common malignant tumors in women all over the world. Many of these women resist the common treatments. Therefore, it is important to find new products to increase the efficacy of the treatment process. Legume beans, with their various pharmacological properties, can be regarded as a sensitizer when they are combined with radiation. The present study strove to survey the radio-sensitivity effect of proteins isolated from mung bean aqueous extract on the human breast adenocarcinoma cell line (MCF-7), human cervical cancer cells (Hela) and the human dermal fibroblast cell line.

Materials and methods: The mung bean aqueous extract was partially purified by ammonium sulfate. At first, various concentrations of the extracts were used to evaluate the inhibitory activity by MTT cell proliferation assay.

Results: The results showed that MCF-7 cells and Hela cells were inhibited by an IC₅₀ value of less than 250 and 411?µg/ml, respectively, but it proved to have a proliferation effect on the fibroblast cells. Then, the cells were incubated with 250?µg/ml extract and exposed to 2, 4, and 6?Gy of X-ray radiation. The percentage of the cell survival was investigated through MTT and the clonogenic assay. Apoptosis was measured using acridine orange/ethidium bromide staining. The results demonstrated that the treated MCF-7 cells and Hela cells had significant radio-sensitivity compared with the results of the control group in radiation dose manner in all MTT, clonogenic, and apoptosis assays. In contrast, the treated fibroblast showed a protective effect against radiation.

Conclusion: The results suggest that mung bean proteins have the capacity to be regarded as a radio-sensitizer for breast cancer. Our results also indicated that it could be worth to investigate on mung bean proteins further and they should be tested in animal models for being treated in radiotherapy.     

Palmatine hydrochloride mediated photodynamic inactivation of breast cancer MCF-7 cells: Effectiveness and mechanism of action

Breast cancer is one of the commonest malignant tumors threatening to women. The present study aims to investigate the effect of photodynamic action of palmatine hydrochloride (PaH), a naturally occurring photosensitizer isolated from traditional Chinese medicine (TCM), on apoptosis of breast cancer cells. Firstly, cellular uptake of PaH in MCF-7 cells was measured and the cytotoxicity of PaH itself on breast cancer MCF-7 cells was estimated using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Subcellular localization of PaH in MCF-7 cells was observed using confocal laser scanning microscopy (CLSM). For photodynamic treatment, MCF-7 cells were incubated with PaH and then irradiated by visible light (470 nm) from a LED light source. Photocytotoxicity was investigated 24 h after photodynamic treatment using MTT assay. Cell apoptosis was analyzed 18 h after photodynamic treatment using flow cytometry with Annexin V/PI staining. Nuclear was stained using Hoechst 33342 and observed under a fluorescence microscope. Intracellular production of reactive oxygen species (ROS) was studied by measuring the fluorescence of 2, 7-dichlorofluorescein (DCF) using a flow cytometry. Results showed that PaH treatment alone had no or minimum cytotoxicity to MCF-7 cells after incubation for 24 h in the dark. After incubation for 40 min, the cellular uptake of PaH reached to the maximum, and PaH mainly located in mitochondria and endoplasmic reticulum of MCF-7 cells. Photodynamic treatment of PaH demonstrated a significant photocytotoxicity on MCF-7 cells, induced remarkable cell apoptosis and significantly increased intracellular ROS level. Our findings demonstrated that PaH as a naturally occurring photosensitizer induced cell apoptosis and significantly killed MCF-7 cells.     

低剂量辐射对CIK细胞杀伤乳腺癌细胞作用的影响

目的 研究低剂量辐射对CIK细胞杀伤乳腺癌细胞MCF-7和SK-BR-3作用的影响,探讨其可能的作用机制。方法 取健康人外周血浓缩白细胞,常规分离出单个核细胞,加入不同的细胞因子培养7 d,诱导DC与CIK细胞,并将其按1 :5的比例共同培养7 d获得DC-CIK细胞。DC-CIK及CIK细胞分别给予40、80、120 mGy X射线照射,流式细胞仪检测细胞表型变化,运用CCK-8的方法检测其对乳腺癌细胞的杀伤作用。结果 DC-CIK组对乳腺癌细胞SK-BR-3较CIK组在不同效靶比均有更强的杀伤活性(t=3.63、5.62、8.21、5.49,P<0.05)。与CIK 0 mGy组相比,CIK细胞联合40、80、120 mGy低剂量辐射对乳腺癌细胞MCF-7和SK-BR-3的杀伤活性明显增高,而DC-CIK细胞联合低剂量辐射组与DC-CIK 0 mGy组相比,差异无统计学意义。结论 低剂量辐射对CIK细胞杀伤乳腺癌细胞有协同作用。