Synergy between Laminin-Derived Elastin-like Polypeptides (LELPs) Optimizes Cell Spreading.

A major component of the extracellular matrix (ECM), laminins, modulates cells via diverse receptors. Their fragments have emerging utility as components of "ECM-mimetics" optimized to promote cell-based therapies. Recently, we reported that a bioactive laminin peptide known as A99 enhanced cell binding and spreading via fusion to an elastin-like polypeptide (ELP). The ELP "handle" serves as a rapid, noncovalent strategy to concentrate bioactive peptide mixtures onto a surface. We now report that this strategy can be further generalized across an expanded panel of additional laminin-derived elastin-like polypeptides (LELPs). A99 (AGTFALRGDNPQG), A2G80 (VQLRNGFPYFSY), AG73 (RKRLQVQLSIRT), and EF1m (LQLQEGRLHFMFD) all promote cell spreading while showing morphologically distinct F-actin formation. Equimolar mixtures of A99:A2G80-LELPs have synergistic effects on adhesion and spreading. Finally, three of these ECM-mimetics promote the neurite outgrowth of PC-12 cells. The evidence presented here demonstrates the potential of ELPs to deposit ECM-mimetics with applications in regenerative medicine, cell therapy, and tissue engineering.

Association of Antipsychotic Drugs with the Risk of Recurrent Venous Thromboembolism: A Retrospective Study of Data from a Japanese Inpatient Database.

BACKGROUND: Approximately a decade has passed since the addition of venous thromboembolism to the list of significant adverse reactions of antipsychotic drugs. However, only a few studies have investigated the relationship between antipsychotic use and venous thromboembolism in the Japanese population. PURPOSE: We aimed to evaluate the risk of recurrent venous thromboembolism in users of antipsychotic drugs and update the evidence on venous thromboembolism in the Japanese population. METHODS: A cross-sectional retrospective analysis of data from a large Japanese claims database, managed by Medical Data Vision Co. Ltd., was conducted. Adult patients who experienced venous thromboembolism between October 2014 and September 2018 in acute care hospitals were identified. The risk of recurrent venous thromboembolism was evaluated with logistic regression using demographic variables. The data of patients using antipsychotic drugs within specific therapeutic classes were also evaluated. RESULTS: We included 8960 patients (mean age, 69 years; 59.2% female). Recurrent venous thromboembolism was observed in 686 patients (7.7%). The risk of recurrent venous thromboembolism was significantly higher in younger patients [< 65 years: reference; 65-74 years: odds ratio (OR) 0.81, 95% confidence interval (CI) 0.66-0.99, p = 0.04; ≥ 75 years: OR 0.77, 95% CI 0.64-0.94, p = 0.01], those with history of surgery (OR 1.39, 95% CI 1.18-1.65, p = 0.01), and anticoagulant users (OR 2.25, 95% CI 1.46-3.48, p = 0.01) and was significantly lower in the presence of comorbidities (OR 0.68, 95% CI 0.58-0.81, p< 0.01) and fractures (OR 0.49, 95% CI 0.26-0.94, p = 0.03). Long-term antipsychotic drug prescriptions (> 14 days) were associated with a higher risk of venous thromboembolism than short-term prescriptions (≤ 14 days) (OR 1.56, 95% CI 1.04-2.34, p = 0.03). CONCLUSIONS: In patients with a history of venous thromboembolism, particular attention should be paid to recurrence in younger patients. If antipsychotic drugs are prescribed for > 14 days to patients with a history of venous thromboembolism, they should be administered carefully, guided by reported findings. Further evaluations using different databases or populations are required to generalize the findings of this study.

Bevacizumab-induced proteinuria and its association with antihypertensive drugs: A retrospective cohort study using a Japanese administrative database.

Proteinuria is a major side-effect of the anti-tumor drug bevacizumab, although its incidence and risk factors in the real world are still unclear. Although renin-angiotensin-aldosterone system inhibitors are used clinically to prevent proteinuria, their efficacy remains unclear. The aim of the present study was to reveal the incidence and risk factors of bevacizumab-induced proteinuria and examine the effectiveness of antihypertensive drugs in preventing proteinuria. We conducted a retrospective cohort study using the National Hospital Organization Clinical Data Archives and Medical Information Analysis Databank. Hospitalized patients who received bevacizumab between January 1, 2016, and June 30, 2019, were included. The study outcome was proteinuria within 12 months of bevacizumab administration. Patient characteristics, laboratory tests, and medications were compared between patients with and without proteinuria using multivariable logistic regression analysis. Among the 2,458 patients, 27% developed proteinuria after bevacizumab administration. Nursing dependence (odds ratio [OR], 2.40; 95% confidence interval [CI], 1.89-3.05; P<0.001) and systolic blood pressure ≥140 mmHg (OR, 1.44; 95% CI, 1.17-1.79; P<0.001) were identified as risk factors. Patients with an estimated glomerular filtration rate (eGFR) of 60-89, 45-59, and <45 mL/min/1.73 m2 had 29.7%, 76.8%, and 66.0% higher odds of proteinuria, respectively, than those with an eGFR ≥90 mL/min/1.73 m2. No significant relationship was observed between antihypertensive drugs and the occurrence of proteinuria. More patients may suffer from proteinuria after bevacizumab administration than previously reported. Nursing dependence and systolic blood pressure are predictive risk factors for bevacizumab-induced proteinuria. Patients at risk of proteinuria should be closely monitored.

Effect of prednisolone and saireito co-administration on T-cells of rheumatoid arthritis patients.

OBJECTIVES: Rheumatoid arthritis (RA) is an autoimmune disease. Methotrexate (MTX) and prednisolone (PSL) are used in combination for severe RA therapy. However, it can increase the risk of osteoporosis and osteonecrosis. Saireito (114) can be used to reduce PSL dose owing to its immunosuppressive effects. However, the effect of combination therapy of PSL+114 on the immune system of RA patients remains unknown. This study compared the effect of PSL alone and PSL+114 on peripheral blood mononuclear cell (PBMC) proliferation, T-cell subsets, and cytokine production in adult RA patients receiving MTX monotherapy. METHODS: We isolated PBMCs from 14 consenting RA patients, and cultured them with PSL (0.0001-1.0 µM) in combination with or without 114 (300 µg/mL) for 96 h in the presence of concanavalin A. We measured the proliferation rates of PBMC, proportions of CD4+, CD8+, and CD4+CD25+Foxp3+T-cells (induced T-regulatory cells), and concentrations of interferon-γ, interleukin (IL)-6, IL-10, IL-17A, and tumour necrosis factor in the culture supernatant. RESULTS: Compared to the blank, the PBMC proliferation rate significantly decreased at a reduced PSL concentration after 114 administration. The 50% inhibition concentration was 0.43 µM PSL for the PSL-only group as compared to 0.29 µM PSL for the PSL+114 co-administration group. The PSL+114 co-administration group had a significantly higher concentration of IL-6 compared to the PSL-only group. CONCLUSIONS: The use of 114 in combination with low-concentration PSL intensified its immunosuppressive effect. However, the concentration of IL-6 was elevated in the co-administration group, suggesting exacerbation of RA activity.

[Analysis the Selection of Opioid Analgesics with Mild and Moderate Cancer Pain Patients for Opioid Naïve].

In 2018, the World Health Organization revised its cancer pain therapy, abolishing the three-step pain relief ladder and recommending the use of opioid analgesics(OA)according to the pain intensity. Of opioid naive patients who were admitted to Chibaken Saiseikai Narashino Hospital from July 2015 to June 2017, treatment with weak OA was initiated in 13 patients(WOA group)and low-dose strong OA in 12 patients(SOA group). The numerical rating scale values immediately before the start of OA and 3, 7 and 14 days later were not significantly different between the 2 groups. As for adverse events, the frequency of occurrence(p=0.01)and the prolongation of the last onset date(p=0.02)were significant in the WOA group for constipation. When the factors related to OA selection were analyzed using logistic regression analysis, there was no significance. We reported the analysis results regarding OA selection in OA naive patients.

Salivary inflammatory mediators as biomarkers for oral mucositis and oral mucosal dryness in cancer patients: A pilot study.

Oral mucositis (OM) is a common side effect in patients with cancer receiving chemotherapy and radiotherapy; however, no salivary mediator is known to be associated with OM. We aimed to determine candidate salivary inflammatory mediators potentially associated with OM in patients with cancer. To this end, we compared the relationships between OM grade, oral mucosal dryness, and inflammatory mediators (Interleukin (IL)-1ß, IL-6, IL-10, IL-12p70, tumor necrosis factor (TNF), prostaglandin E2, and vascular endothelial growth factor) in patients with cancer and in healthy volunteers (HV). We collected saliva samples from 18 patients with cancer according to the following schedule: 1) within 14 days of treatment initiation, 2) within 3 days of OM occurrence, 3) when OM was improved or got worsened, and 4) within 7 days after chemotherapy completion. The oral care support team determined the OM grade at each sample collection point based on CTCAE version 5.0. Salivary inflammatory mediator concentrations were detected using cytometric bead array or enzyme-linked immunoassay. We compared oral mucosal dryness in pre- and post-index patients with cancer to that in HV (n = 33) using an oral moisture-checking device. Fourteen of eighteen patients experienced OM (four, grade 3 OM; four, grade 2 OM; six, grade 1 OM). IL-6, IL-10, and TNF salivary concentrations were significantly increased in the post-index group compared to those in the pre-index group (p = 0.0002, p = 0.0364, and p = 0.0160, respectively). Additionally, salivary IL-6, IL-10, and TNF levels were significantly higher in the post-index group than in the HV group (p < 0.0001, p < 0.05, and p < 0.05, respectively). Significant positive correlations were observed between OM grade and salivary IL-6, IL-10, and TNF levels (p = 0.0004, r = 0.4939; p = 0.0171, r = 0.3394; and p = 0007, r = 0.4662, respectively). Oral mucosal dryness was significantly higher in the HV than in the pre- and post-index groups (p < 0.001). Our findings suggest that salivary IL-6, IL-10, and TNF levels may be used as biomarkers for OM occurrence and grade in patients with cancer. Furthermore, monitoring oral mucosal dryness and managing oral hygiene before cancer treatment is essential.

Saireito (114) Increases IC50 and Changes T-Cell Phenotype When Used in Combination with Prednisolone Therapy in Human Peripheral Blood Mononuclear Cells.

Prednisolone (PSL), a type of corticosteroid used to treat autoimmune diseases, can increase the risk of infection and osteoporosis. Saireito (114), a Kampo medicine, has an immunosuppressive effect; with its use, the dose of steroids can be reduced. However, its mechanism when used with PSL is still unclear. We used peripheral blood mononuclear cells (PBMCs) from healthy adults to examine the effect of 114 and PSL treatment on PBMC proliferation, T-cell subsets, and cytokine production. PBMCs were cotreated with concanavalin A and 300 µM 114 (either Tsumura & Co. (TJ) or Kracie Holdings (KR)) and 0.0001-1.0 µM PSL for 96 h to create the T-cell mitogen. We then measured the PBMC proliferation; ratio of CD4+ T cells, CD8+ T cells, and T-follicular helper (Tfh) cells; and concentration of cytokines (TNF, IFN-γ, IL-6, IL-10, IL-17A, and IL-21). The proliferation of PBMCs was dose dependently suppressed in both the PSL and PSL + 114 groups (p < 0.05). Combination therapy increased the IC50 in the PSL group (0.0947 µM) by 2.02 and 1.64-fold in the PSL + TJ114 and PSL + KR114 groups, respectively. Both the PSL + 114 groups had an increased ratio of CD4+ T cells compared to the PSL group, with no effect on the ratio of CD8+ T and Tfh cells. Furthermore, the PSL + 114 groups showed increased IL-6 and IL-10 compared to the PSL monotherapy group, although the difference was not significant. There was no significant difference in the TNF, IFN-γ, IL-17A, and IL-21 concentrations between the PSL and PSL + 114 groups. The elevated IC50 with 114 cotreatment suggests diminished immunosuppressive action. Moreover, increased cytokine production by Th2 with 114 cotreatment suggests a restoration of T-cell balance in Th1-mediated autoimmune diseases. However, increased IL-6 suggests potential exacerbation of IL-6-mediated diseases, such as rheumatoid arthritis. Therefore, it is necessary to monitor these clinical parameters when using 114 in combination with PSL.

Immunological Differences in Human Peripheral Blood Mononuclear Cells Treated with Traditional Japanese Herbal Medicines Hochuekkito, Juzentaihoto, and Ninjin'yoeito from Different Pharmaceutical Companies.

Hochuekkito (HET), Juzentaihoto (JTT), and Ninjin'yoeito (NYT) have been used as Hozai, a group of traditional Japanese herbal medicines, to treat physically and mentally weak cancer patients. Their compositions are quite different, and Japanese pharmaceutical companies have been using different types or quantities of herbs for formulations with the same name. Here, we compared the immunological differences between HET, JTT, and NYT with respect to the induced T cell subsets and cytokines. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers and treated with 0 (control), 25, 50, 100, 200, or 400 µg/mL HET, JTT, or NYT (manufactured by Tsumura [TJ], Kracie [KR], and Kotaro [KO]). PBMC proliferation, CD4+ T cell, CD8+ T cell, and regulatory T cell (Treg) proportions and interleukin (IL) concentrations (IL-6, IL-10, IL-17A, interferon-γ, tumor necrosis factor-α, and transforming growth factor (TGF)-ß) secreted by PBMCs were measured using Cell Counting Kit-8 or flow cytometry bead analysis. PBMC proliferation and CD4+ T cell percentages were similar in the HET, JTT, NYT, and control groups; however, the percentage of CD8+ T cells tended to increase after treatments. Tregs were suppressed by HET, JTT, and NYT, and TJ-JTT significantly decreased Treg numbers (compared with control). The concentrations of all cytokines except TGF-ß were increased in a concentration-dependent manner (p < 0.05); particularly, KR-HET induced IL-6 secretion (compared with the control, TJ-HET, and KO-HET; 37-, 7-, and 17-fold, respectively; p < 0.05). The TGF-ß concentration was decreased in a concentration-dependent manner by HET, JTT, and NYT (compared with the control). These results suggest that, compared with TJ-HET and KO-HET, KR-HET should be administered with caution. Although HET, JTT, and NYT belong to the same Hozai group and have the same names among companies, their differing effects on immune activity must be considered and they must be administered with caution.

Cytotoxic Effects of Hellebrigenin and Arenobufagin Against Human Breast Cancer Cells.

Development of new therapeutic strategies for breast cancer is urgently needed due to the sustained emergence of drug resistance, tumor recurrence and metastasis. To gain a novel insight into therapeutic approaches to fight against breast cancer, the cytocidal effects of hellebrigenin (Helle) and arenobufagin (Areno) were investigated in human estrogen receptor (ER)-positive breast cancer cell line MCF-7 and triple-negative breast cancer cell line MDA-MB-231. Helle exhibited more potent cytotoxicity than Areno in both cancer cells, and MCF-7 cells were more susceptible to both drugs in comparison with MDA-MB-231 cells. Apoptotic-like morphological characteristics, along with the downregulation of the expression level of Bcl-2 and Bcl-xL and the upregulation of the expression level of Bad, were observed in Helle-treated MCF-7 cells. Helle also caused the activation of caspase-8, caspase-9, along with the cleavage of poly(ADP-ribose) polymerase in MCF-7 cells. Helle-mediated necrosis-like phenotype, as evidenced by the increased propidium iodide (PI)-positive cells was further observed. G2/M cell cycle arrest was also induced by Helle in the cells. Upregulation of the expression level of p21 and downregulation of the expression level of cyclin D1, cyclin E1, cdc25C and survivin were observed in MCF-7 cells treated with Helle and occurred in parallel with G2/M arrest. Autophagy was triggered in MCF-7 cells and the addition of wortmannin or 3-MA, two well-known autophagy inhibitors, slightly but significantly rescued the cells. Furthermore, similar alterations of some key molecules associated with the aforementioned biological phenomena were observed in MDA-MB-231 cells. Intriguingly, the numbers of PI-positive cells in Helle-treated MCF-7 cells were significantly reduced by wortmannin and 3-MA, respectively. In addition, Helle-triggered G2/M arrest was significantly corrected by wortmannin, suggesting autophagy induction contributed to Helle-induced cytotoxicity of breast cancer cells by modulating necrosis and cell cycle arrest. Collectively, our results suggested potential usefulness of both Helle and Areno in developing therapeutic strategies to treat patients with different types of breast cancer, especially ER-positive breast cancer.

Healthcare utilization associated with adherence to antibiotics for abdominal surgeries in Japan: cross-sectional analysis of administrative database.

BACKGROUND: Since patients receiving surgery may experience surgical site infections, therapeutic guidelines for reducing hospitalization time and cost include appropriate antibiotic use. However, the association between adherence to therapeutic guidelines and healthcare utilization is currently unclear. OBJECTIVES: This study aimed to confirm the positive association between the adherence to guidelines of antibiotic therapy and a reduction in the length of stay and cost of hospitalization, especially considering the high infection rates in abdominal surgery. METHODS: This cross-sectional study used administrative data (diagnosis procedure combination data) collected using the case-mix system implemented in acute-care hospitals in Japan. We assessed the length of hospital stay and cost of hospitalization for patients who received prophylactic antibiotic for abdominal surgeries consistent with therapeutic guidelines. The data of patients aged 15 years or older who received appendectomy, laparoscopic cholecystectomy or inguinal hernia repair were extracted. The appropriateness of antibiotic prophylaxis was evaluated in terms of the Japanese guidelines for antibiotic selection and treatment duration. To assess the mean difference in antibiotic costs and length of stay, we performed the propensity score matching by confounding factors. Furthermore, we assessed the progress in healthcare utilization of this therapy over a decade. RESULTS: Of the 302 233 patients who received single general surgery from April 2014 to March 2016, 198 885 were eligible for analysis after applying the exclusion criteria (143 975 in the adherence and 54 910 in the non-adherence group). Each group comprised 48 439 patients after propensity score matching. Inappropriate antibiotic selection and duration were observed in 9294 (9.8%) and 687 (0.7%) of inguinal hernia repairs, 6431 (25.3%) and 311 (1.2%) of appendectomies and 38 134 (48.5%) and 391 (0.5%) of laparoscopic cholecystectomy cases, respectively. After propensity score matching by operation type, average hospitalization length (6.5 [SD 3.8] and 7.3 [SD 4.8] days) and costs (536 000 [SD 167 000] JPY and 573 000 [SD 213 000] JPY) differed significantly between adherence and non-adherence groups. CONCLUSION: The results revealed that unnecessary healthcare utilization was associated with failure to adhere to therapeutic guidelines for prophylactic antibiotic therapy in elective general surgeries. We concluded that the progress of reduction in length of hospitalization over the decade was successful. Notably, adherence to treatment duration was better than that was 10 years ago. In this decade, administrators in hospitals have attempted to reduce the duration of hospitalization by developing various clinical pathways for surgical procedures and quality indicators. However, 15 877 patients (8.7%) were prescribed oral antibiotics the day after surgery. These observations should be evaluated further.

[Optimization of Preparation, and Stability of Indomethacin Mouth Wash for Oral Mucositis Caused by Cancer Therapy].

OBJECTIVE: Severe oral mucositis caused by chemo- and radio-therapy is a common adverse event in patients with cancer. In this study, we investigated the development of an indomethacin mouth wash(IM-MW)as a novel approach to treat pain due to oral mucositis. METHODS: We examined the appropriate preparation methods for IM-MW with suitable stability. IM- MW was made from bulk IM, controlled release IM capsules, and IM capsules. Dissolution in water was tested at water temperatures of 70℃, 90℃, and 98℃(n=3), and with a shaking time of 30 or 60s(n=3). We determined the IM concentration in IM-MW by HPLC-UV analysis(n=5)at time points between just after preparation and day 7, to estimate the shelf- life at 4℃ and 25℃. RESULTS: At 70℃, bulk IM did not dissolve, but at 90℃ and 98℃, bulk IM, controlled release IM capsules, and IM capsules all dissolved effectively. Shaking times of 30 and 60s were sufficient to dissolve bulk IM, controlled release IM capsules, and IM capsules. The stability of IM in IM-MW was 98.6±2.8%(bulk), 99.2±6.0%(controlled release capsule), and 98.5±6.0%(capsule)over 7 days at 4℃. However, at 25℃, IM stability in IM-MW decreased to 95.3±1.8% (bulk), 86.1±4.8%(controlled release capsule), and 83.6±1.6%(capsule). CONCLUSION: In this study, we identified the most suitable method for the preparation of IM-MW(90℃, shaking time of over 30s). IM-MW was stable when stored at 4℃ for at least 7 days after preparation.

Potent antitumor activity of cepharanthine against triple-negative breast cancer spheroids compared with tetrandrine.

Cepharanthine (CEP) is a bis-bynzelisoquinoline alkaloid from the same class as the anticancer agent tetrandrine (TET). However, the effects of CEP against breast cancer have not been extensively studied, despite its long therapeutic history with low toxicity against other types of cancer. 3D culture systems more accurately mimic the human body and address the limitations of determining drug effectiveness compared with 2D culture systems. In the present study, the antitumor activities of TET and CEP were compared in 3D culture systems in triple-negative breast cancer (TNBC) MDA-MB-231 and estrogen receptor-positive breast cancer MCF-7 cell lines. Cell viability, apoptosis and cytotoxicity assays were performed to determine the total number of live or dead cells, the IC50 values, the number of apoptotic cells and spheroid roundness. Viability suppression of MDA-MB-231 cells was significantly greater with both TET and CEP compared with that of MCF-7 cells, and the roundness of MDA-MB-231 spheroids treated with CEP was decreased significantly compared with that of spheroid treated with TET. Cytoplasmic shrinkage in each cell line significantly increased with the treatment of TET compared with the control; however, this effect was stronger with CEP. The ratio of dead/live cells in each cell line treated with TET and CEP increased in a dose-dependent manner. Overall, the present study demonstrated that CEP had greater cell toxicity in 3D spheroids of breast cancer cells compared with TET, suggesting that CEP may have a stronger antitumor activity on TNBC spheroids compared with TET.

JNK and Autophagy Independently Contributed to Cytotoxicity of Arsenite combined With Tetrandrine via Modulating Cell Cycle Progression in Human Breast Cancer Cells.

Novel therapeutic strategies for breast cancer are urgently needed due to the sustained development of drug resistance and tumor recurrence. Trivalent arsenic derivative (arsenite, AsIII) has been reported to induce cytotoxicity in breast cancer cells. We recently demonstrated that AsIII plus tetrandrine (Tetra), a Chinese plant-derived alkaloid, exerted potent antitumor activity against human breast cancer cells, however, the underlying mechanisms for their action have not been well defined. In order to provide fundamental insights for understanding the action of AsIII plus Tetra, the effects of the combined regimen on two breast cancer cell lines T47D and MDA-MB-231 were evaluated. Compared to T47D cells, MDA-MB-231 cells were much more susceptible to the synergistic cytotoxic effects of AsIII and Tetra. Besides the induction of apoptotic/necrotic cell death, S-phase arrest and autophagic cell death were also observed in MDA-MB-231 cells. Exposure of MDA-MB-231 cells to AsIII and Tetra caused the activation of MAPKs. Cytotoxicity of the combined regimen in MDA-MB-231 cell was significantly abrogated by SP600125, a potent c-Jun N-terminal kinase (JNK) inhibitor. However, similar abrogation was not caused by p38 and ERK inhibitors. The addition of either autophagy inhibitors (3-methyladenine or wortmannin) or SP600125 corrected the combined regimen-triggered S-phase arrest, whereas had little effect on the apoptosis/necrosis induction in the cells. Surprisingly, SP600125NC, a negative control for SP600125, significantly strengthened S-phase arrest and the cytotoxicity induced by the combined regimen. The addition of SP600125 did not alter autophagy induction. In conclusion, the cytotoxicity of AsIII combined with Tetra was attributed to the induction of S-phase arrest, apoptotic/necrotic and autophagic cell death. The enhanced cytotoxicity of the two drugs by SP600125NC might be explained by its capability to strengthen S-phase arrest. Our results suggested that JNK and autophagy independently contributed to the cytotoxicity via modulating cell cycle progression. The study further provides fundamental insights for the development of AsIII in combination with Tetra for patients with different types of breast cancer.

Arsenic Disulfide Combined with L-Buthionine-(S, R)-Sulfoximine Induces Synergistic Antitumor Effects in Two-Dimensional and Three-Dimensional Models of MCF-7 Breast Carcinoma Cells.

Three-dimensionally (3D) cultured tumor cells (spheroids) exhibit more resistance to therapeutic agents than the cells cultured in traditional two-dimensional (2D) system (monolayers). We previously demonstrated that arsenic disulfide (As2S2) exerted significant anticancer efficacies in both 2D- and 3D-cultured MCF-7 cells, whereas 3D spheroids were shown to be resistant to the As2S2 treatment. L-buthionine-(S, R)-sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, has been regarded to be a potent candidate for combinatorial treatment due to its GSH modulation function. In the present study, we introduced BSO in combination with As2S2 at a low concentration to investigate the possible enhancing anticancer efficacy by the combinatorial treatment on 2D- and 3D-cultured MCF-7 cells. Our results presented for the first time that the combination of As2S2 and BSO exerted potent anticancer synergism in both MCF-7 monolayers and spheroids. The IC50 values of As2S2 in combinatorial treatment were significantly lower than those in treatment of As2S2 alone in both 2D- and 3D-cultured MCF-7 cells (P<0.01, respectively). In addition, augmented induction of apoptosis and enhanced cell cycle arrest along with the regulation of apoptosis- and cell cycle-related proteins, as well as synergistic inhibitions of PI3K/Akt signals, were also observed following co-treatment of As2S2 and BSO. Notably, the combinatorial treatment significantly decreased the cellular GSH levels in both 2D- and 3D-cultured MCF-7 cells in comparison with each agent alone (P<0.05 in each). Our results suggest that the combinatorial treatment with As2S2 and BSO could be a promising novel strategy to reverse arsenic resistance in human breast cancer.

Differentiation induction of human breast cancer cells by arsenite in combination with tetrandrine.

BACKGROUND: To provide novel insight into the development of new therapeutic strategies to combat breast cancer, differentiation-inducing activity of clinically achievable concentrations of arsenite (AsIII) and tetrandrine (Tetra) was investigated in breast cancer cell lines MDA-MB-231 and MCF-7. METHODS: Differentiation induction of cancer cells was analyzed by flow cytometer. Alterations of genes related to differentiation, and proliferation of human normal peripheral blood mononuclear cells (PBMCs) were analyzed using western blotting and cell viability assay, respectively. RESULTS: Exposure to Tetra alone or in combination with AsIII induced differentiation of both cells characterized by upregulation of ICAM-1, downregulation of Her2/neu. In comparison with MCF-7, the combination of lower concentrations of AsIII and Tetra induced differentiation of MDA-MB-231, indicating that MDA-MB-231 cells were highly susceptible to differentiation. The differentiation occurred in parallel with activation of Erk signaling pathway, and was abolished by PD98059, a potent Erk inhibitor. Consistent with in vitro experimental results, the upregulation of ICAM-1 and the activation of Erk signaling pathway were also observed in MDA-MB-231 breast tumors in xenograft mouse obtained from our previous study. No obvious proliferation inhibition of PBMCs was observed following the exposure to AsIII combined with Tetra at the concentrations capable of inducing differentiation of MDA-MB-231 cells. CONCLUSION: The Erk signaling pathway may be crucially involved in the differentiation induction of breast cancer cells in vitro and in vivo. Collectively, our results suggest that the combination can probably serve as promising candidates for the development of novel therapeutic approaches for different types of breast cancer.

Arsenic disulfide­induced apoptosis and its potential mechanism in two­ and three­dimensionally cultured human breast cancer MCF­7 cells.

In China, arsenic disulfide (As2S2) has been used for the treatment of hematological malignancies. The present study aimed to evaluate the effects of As2S2 on the human breast cancer MCF­7 cell line cultured in both two­dimensional (2D) monolayers and three­dimensional (3D) spheroids to explore its therapeutic potential in breast cancer treatment. Cellular viability and the induction of apoptosis were examined with a cell counting kit­8 (CCK­8) assay and flow cytometric analysis, respectively. Alterations in the expression levels of apoptosis­associated proteins, including Bcl­2­associated X protein (Bax), B­cell lymphoma 2 (Bcl­2), p53, and caspase­7, as well as the cell survival­associated proteins, phosphatidylinositol 3­kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR), were assessed by western blotting. Although a dose­dependent reduction in cell viability, which occurred in association with the induction of apoptosis triggered by the addition of 2­24 µM As2S2, was observed in both 2D­ and 3D­culture systems, 3D spheroids were less sensitive to the cytotoxic effect of As2S2 compared with the 2D cultured cells. A significant increase in the expression levels of Bax, p53, and caspase­7 was observed in treated 2D­cultured cells, whereas a similar increase in the expression levels of Bax was only confirmed in treated 3D spheroids, although there was a trend towards the increased expression of p53 and caspase­7 in the 3D spheroids. These results suggested that these molecules are closely associated with As2S2­mediated cytotoxicity in the two culture systems, and further suggested that the difference in the sensitivity to As2S2 between 2D monolayers and 3D spheroids may be attributed to the differential alterations in the expression levels of proteins associated with cell mortality. Significant downregulation of the expression levels of Bcl­2, PI3K, Akt and mTOR was observed in the two culture systems. Taken together, the results of the present study demonstrated that As2S2 inhibits cell viability and induces apoptosis in both 2D­ and 3D­ cultured MCF­7 cells, which may be associated with activation of the pro­apoptotic pathway and the inhibition of pro­survival signaling. These results have provided novel insights into clinical applications of As2S2 in the treatment of patients with breast cancer.

Chemo-sensitivity of Two-dimensional Monolayer and Three-dimensional Spheroid of Breast Cancer MCF-7 Cells to Daunorubicin, Docetaxel, and Arsenic Disulfide.

BACKGROUND/AIM: Chemo-sensitivity of two-dimensional (2D) monolayers and three-dimensional (3D) spheroids of human breast cancer MCF-7 cells were investigated. MATERIALS AND METHODS: MCF-7 cells were cultured in monolayers or spheroids established using a thermo-reversible gelatin polymer, in the presence of daunorubicin, docetaxel, or As2S2 Cell proliferation was examined by a Cell Counting Kit-8 assay. RESULTS: Daunorubicin, docetaxel, and As2S2 dose-dependently decreased the MCF-7 cell proliferation in both 2D- and 3D-culture systems. The 3D spheroids were less sensitive to these agents than the 2D cultured cells. Verapamil, an inhibitor of P-glycoprotein, partially enhanced the antiproliferative effects of the agents. DL-buthionine-(S, R)-sulfoximine significantly increased (p<0.05), while N-acetyl-L-cysteine significantly inhibited the antiproliferative effects of As2S2 (p<0.003). CONCLUSION: The 3D spheroids showed less sensitivity to the antiprolliferative efficacies of anticancer agents than the 2D cultured cells. P-Glycoprotein is suggested to be partially implicated in drug resistance. Reduction of cellular glutathione level enhanced the As2S2 cytotoxicity.

Effects of arsenic disulfide on proliferation, cytokine production, and frequencies of CD4(+), CD8(+), and regulatory T cells in mitogen-activated human peripheral blood mononuclear cells.

Influence of arsenic disulfide (As2S2) on human immune cells has little been investigated. Effects of As2S2 on proliferation, cytokine production, and frequencies of CD4(+) T, CD8(+) T and CD4(+)CD25(+)Foxp3(+) regulatory T cells in mitogen-activated human peripheral blood mononuclear cells were examined. Anti-proliferative effects of As2S2 on peripheral blood mononuclear cells activated by T-cell mitogen were assessed by a colorimetric assay. Cytokine concentrations in the culture medium were measured with beads-array procedures followed by flow cytometry. CD4(+) T cells, CD8(+) T cells and CD4(+)CD25(+)Foxp3(+) regulatory T cells were stained with fluorescence-labeled specific antibodies followed by flow cytometry analysis. As2S2 at 1-10µM significantly suppressed mitogen-activated proliferation of peripheral blood mononuclear cells (p<0.05). As2S2 at 10µM inhibited production of IL-6, -10, -17A, tumor necrosis factor-α, and interferon-γ from the activated peripheral blood mononuclear cells, though the effects were not statistically significant. As2S2 at 10µM significantly suppressed the frequencies of CD4(+) T and CD8(+) T cells (p<0.05), whereas significantly enhanced the frequency of CD4(+)CD25(+)Foxp3(+) regulatory T cells (p<0.05). The data suggest that As2S2 attenuates T cell-mediated immunity by not only suppressing the proliferation of T cells and cytokine release but also increasing the frequency of regulatory T cells. T cell-mediated autoimmunity contributes to bone marrow failure in myelodysplastic syndrome (MDS), and thus the above As2S2 effects are beneficial for the treatment of MDS patients.

Characterization and Clinical Implication of Th1/Th2/Th17 Cytokines Produced from Three-Dimensionally Cultured Tumor Tissues Resected from Breast Cancer Patients.

OBJECTIVES: Several cytokines secreted from breast cancer tissues are suggested to be related to disease prognosis. We examined Th1/Th2/Th17 cytokines produced from three-dimensionally cultured breast cancer tissues and related them with patient clinical profiles. METHODS: 21 tumor tissues and 9 normal tissues surgically resected from breast cancer patients were cultured in thermoreversible gelatin polymer-containing medium. Tissue growth and Th1/Th2/Th17 cytokine concentrations in the culture medium were analyzed and were related with hormone receptor expressions and patient clinical profiles. RESULTS: IL-6 and IL-10 were expressed highly in culture medium of both cancer and normal tissues. However, IFN-γ, TNF-α, IL-2, and IL-17A were not detected in the supernatant of the three-dimensionally cultured normal mammary gland and are seemed to be specific to breast cancer tissues. The growth abilities of hormone receptor-negative cancer tissues were significantly higher than those of receptor-positive tissues (P=0.0383). Cancer tissues of stage ≥IIB patients expressed significantly higher TNF-α levels as compared with those of patients with stage