And Doxorubicin Research Articles

Cinobufagin inhibits tumor progression and reduces doxorubicin resistance by enhancing FOXO1-mediated transcription of FCGBP in osteosarcoma

Ethnopharmacological relevanceCinobufagin (Huachansu), an aqueous extract from the dried skin of the toad Bufo bufo gargarizans Cantor (frog skin), is a biologically active ingredient of a traditional Chinese medicine cinobufacini that can treat multiple bone pathological conditions such as bone pain, bone tumors, and osteosarcoma. Aim of the studyThe study aimed to explore the roles and molecular mechanisms of cinobufagin underlying osteosarcoma development and doxorubicin (ADR) resistance. Materials and methodsCell viability, migration, and invasion were examined by CCK-8, wound healing, and Transwell invasion assays, respectively. RNA sequencing analysis was performed in MNNG/HOS cells treated with or without cinobufagin. The relationships of cinobufagin, forkhead box O1 (FOXO1), and Fc fragment of IgG binding protein (FCGBP) were examined by luciferase reporter, immunofluorescence (IF), RT-qPCR, and chromatin immunoprecipitation (ChIP) assays together with weighted gene co-expression network analysis (WGCNA) analysis. Epithelial-mesenchymal transition (EMT) marker levels were examined through the Western blot assay. The function and molecular basis of cinobufagin in osteosarcoma were further investigated by mouse xenograft experiments. ResultsCinobufagin reduced cell viability, weakened ADR resistance, and inhibited cell migration/invasion/EMT in osteosarcoma cells. Cinobufagin enhanced FOXO1-mediated transcription of downstream genes including FCGBP. FCGBP knockdown partly abrogated the effect of cinobufagin on osteosarcoma cell development. Cinobufagin inhibited the growth of mouse osteosarcoma xenografts in vivo. Cinobufagin reduced the expression of Ki-67 and MMP9 and facilitated caspase-3 expression in osteosarcoma xenografts. ConclusionCinobufagin suppressed tumor progression and reduced ADR resistance by potentiating FOXO1-mediated transcription of FCGBP in osteosarcoma.

Follicular Lymphoma Response with CDK4/6 Inhibitor(Palbociclib) Utilized in the Setting of Metastatic Breast Cancer

INTRODUCTIONHere we report our clinical observation and first objective evidence of partial response seen in follicular lymphoma from the use of drug (palbociclib) that inhibits cyclin D-dependent kinases (CDK) CDK4 and CDK6. CASE HISTORYA 66 year old female with metastatic breast cancer in the bones and relapsing nodal follicular lymphoma (FL) was initiated on Palbocilib and fulvestrant for her breast cancer.The details of her Non-Hodgkin's lymphoma (NHL) history include her initial presentation in 2005 with large cell lymphoma. She underwent treatment with four cycles of R-CHOP and four cycles of RICE (dose of anthracycline was limited given prior exposure of doxorubicin administered for breast cancer). She achieved complete remission. The patient later relapsed with follicular lymphoma grade I/II in 2011 requiring treatment with six cycles BR(bendamustine, rituximab) followed by two years of R-maintenance. Her treatment completed in 2013. She remained in remission for the next two years and relapsed again in late 2015 with right breast mass and multiple lymph nodes above and below the diaphragm. Biopsy of breast mass and right axillary lymph nodes showed FL Grade IIIa.CD10+, Bcl-2+, Bcl-6+ and Cyclin D1 negative.The details of breast cancer include the initial diagnosis in 2003 stage T2N1 ER/PR positive (estrogen, progesterone), Her-2 was negative. She underwent treatment with lumpectomy, sentinel lymph node biopsy and axillary lymph node dissection. She also received Cyclophosphamide and doxorubicin (AC) for four cycles followed by paclitaxel for four cycles and five years of anastrazole. Later in 2013 as patient completed R-maintenance for the follicular lymphoma she presented with pathological fracture of T8 vertebral body. Biopsy confirmed metastatic breast cancer. CAT-scans showed extensive sclerotic bony metastasis consistent with metastatic breast cancer. The patient received hormonal therapy letrozole and zoledronic acid. The patient responded very well with decrease in breast tumor marker (CA 15-3) and improvement of skeletal pain.The patient progressed two years later in late 2015 with CAT-scan showing progression of sclerotic bone lesions with multiple new foci noted. The imaging also showed multiple areas of lymph nodes above and below diaphragm. Patient also developed palpable right breast mass 3cm and PET-scan showed multiple FDG-avid lymph nodes including bony sclerotic PET-avid lesion. The biopsy of breast mass and axillary lymph nodes showed FL. The radiological progression was also seen in the previously biopsy proven bony sclerotic lesion showing breast cancer. The plan was made to pursue treatment with breast cancer as patient had significant skeletal pain along with once again rise in tumor marker CA 15-3.Patient was initiated treatment on palbocilib and fulvestrant in early 2016. Three months later right breast nodal mass and axillary lymph node was not palpable. PET-scan showed decrease in the size and metabolic activity. Most of the lymph nodes showed shrinkage in size as well as SUV consistent with partial response. With respect to metastatic breast cancer tumor markers normalized and sclerotic foci in the bones also showed radiological improvement.To date patient continues to do well with respect to both malignancies. She has not required any treatment for the FL and does appear to maintain partial response from CDK4/6 inhibition. CONCLUSIONFL is a common and incurable form of NHL characterized by the translocation t (14;18) that results in increased expression of the anti-apoptotic BCL2 protein. Exactly what drives expansion of the malignant B-cells in FL is not clear. However recurrent genetic lesions targeting cell cycle control in FL have been reported including mutation in CYCLIN D3 gene, chromosomal gains of CDK4, losses of CDKN2a/b and RB1.We have seen clinical and radiological evidence of response with regard to FL while patient was on treatment with CDK4/6 inhibitor for the indication of metastatic breast cancer. Given that now we already have Bcl-2 inhibitor drug venetoclax which is approved for CLL, this early clinical observation leads us to hypothesize that it would be worth exploring the combination of CDK4/6 inhibition with BCl-2 inhibitor in FL for further exploration. DisclosuresNo relevant conflicts of interest to declare.

Reversal effect of quercetin on multidrug resistance via FZD7/β-catenin pathway in hepatocellular carcinoma cells

BackgroundChemotherapy has been widely used to treat cancer, but the appearance of multidrug resistance (MDR) is the biggest obstacle to successful chemotherapy. One of the conventional mechanisms of MDR is overexpression of ATP-binding cassette (ABC) transporters such as P-glycoprotein (P-gp/ABCB1) and multidrug resistance-associated proteins (MRPs/ABCCs) that limits the prolonged and efficient use of chemotherapeutic drugs. To enhance the chemosensitivity of tumor cells, attentions have been focused on effective MDR modulators. PurposeThis study aimed to investigate the reversal effect of quercetin on MDR, and explored its mechanism of action in vitro. Study design/methodsThe effect and mechanism of quercetin on MDR was examined by using MTT assay, flow cytometry, real-time PCR and western blot analysis in human hepatocellular carcinoma cells. ResultsOur data found that the intracellular accumulation of rhodamine-123 (Rh123) and doxorubicin (ADR) were increased, the sensitivity of BEL/5-FU cells to chemotherapeutic drugs were increased, and the expressions of ABCB1, ABCC1 and ABCC2 were all down-regulated, which indicated that the functions and expressions of ABCB1, ABCC1 and ABCC2 efflux pump were inhibited by quercetin treatment. Moreover, the suppression of ABCB1, ABCC1 and ABCC2 by quercetin was dependent on the FZD7 through the Wnt/β-catenin pathway. Further research revealed that reduction of FZD7 by RNA interference (siFZD7) enhanced the sensitivity to chemotherapeutic drugs, increased the cellular accumulation of Rh123 and ADR, and induced inhibitory effects on the expression of FZD7, ABCB1, ABCC1, ABCC2 and β-catenin, similar to quercetin. In the meanwhile, overexpression of FZD7 showed the inversely effect on the expressions. Interesting, it was confirmed that quercetin could inhibit the expression levels of FZD7, ABCB1, ABCC1, ABCC2 and β-catenin in BEL-7402 cells; furthermore, treatment by quercetin combined with siFZD7 in BEL/5-FU cells, the expressions of these genes were effectively decreased in comparison to quercetin combined with siRNA negative control (sncRNA). ConclusionOverall, these data suggested the effectiveness of using quercetin, at least in part, via inhibiting FZD7 to combat chemoresistance and showed that quercetin could be developed into an efficient natural sensitizer for resistant human hepatocellular carcinoma.

Response Rates to Neoadjuvant Cyclophosphamide and Doxorubicin (AC) in Breast Cancer Patients Exhibiting Her-2/Neu (Her-2) Negative, Topoisomerase II (TOPO 2A) Normal, Chromosome 17 (C17) Normal Phenotype.

Abstract Background: Recent reports have suggested that Her-2, TOPO2A and polysomy 17 (C17) status may predict responsiveness of tumors to doxorubicin (DOX) therapy. These studies predominantly evaluated long term survival in the adjuvant setting. In this study we evaluate the actual response rate to neoadjuvant AC as a function of these parameters.Material and method: We identified all breast cancer patients in our registry cohort treated with neoadjuvant DOX and cyclophosphamide only prior to surgery. All had hormone receptor (HR) and Her-2 testing as part of their prior clinical evaluation. Forty patients were found, 33 of whom had sufficient tissue for testing and adequate signal for measurement of TOPO2A and C17. Fluorescence in situ hybridization (FISH) testing was done on deparaffinized tissue sections pretreated using a modification of the vendor's standard protocol, and then incubated with the FDA-approved Vysis PathVysion™ probe set, which includes Spectrum-Green-conjugated probe to the α-satellite DNA located at the centromere of chromosome 17 (17p11.1–q11.1) and a Spectrum Orange-conjugated probe to the TOP2A gene (Abbott Diagnostics, Chicago, IL, USA). Morphometric analysis was performed using a MetaSystems image analysis system, incorporating the Metafer software with extended focus/tile sampling methodology (Meta-Systems, Altlussheim, Germany). Manual counting was performed on all samples in which autofluorescence and/or artifact prevented the counting of sufficient numbers of cells. A ratio > 2.0 is considered positive for TOPO2A amplification. C17 presence was defined as signal of ≥ 2.5.Results: Out of 33 patients tested, one was Topo2A amplified (HR-/Her-2-/C17-, partial responder) and one had polysomy 17 present (HR+/Her-2+/TOPO2A-, partial responder). 17 out of 33 were HR+ and eight were Her-2+. Among the TOPO2A non-amplified and C17 normal patients (n=31), treatment response was pathologically proven complete response (pCR) = 5 (16%), PR = 19 (61%), and NR = 7 (23%). Distribution by marker status and response to therapy for the 31 cases was the following: 1) HR-/Her-2- (n=9), 22% pCR, 56% PR, 22% NR; 2) HR+/Her-2- (n=15), 7% pCR, 67% PR, 27% NR; 3) HR-/Her-2+ (n=6), 33% pCR, 67% PR; 4) EstrogenR+/ProgesteroneR-/Her-2+ (n=1), NR. Among TOPO2A non-amplified and C17 normal patients the majority (77%) had significant responsiveness (CR or PR) to AC. The patients that lacked all three postulated indicators of DOX sensitivity (her-2 negative, TOPO2A normal, C17 normal) still exhibited 13% pCR (3/24) and 63% PR (15/24), with HR/Her-2 neg (triple negative) patients being more responsive than HR+ patients.Discussion: Prior predictive studies have not looked directly at response rates to doxorubicin (DOX). This data suggests that even in patients lacking all three postulated predictors there is significant response to therapy that would be difficult to attribute to cyclophosphamide alone. Patients lacking Her-2, TOPO2A and C17 should not be excluded from receiving DOX-containing regimens, either as primary therapy or as therapy for metastatic disease. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2033.

HER‐2/neu expression as a predictor of response to neoadjuvant docetaxel in patients with operable breast carcinoma

The use of biologic markers to predict response to neoadjuvant chemotherapy may permit tailoring regimens to achieve maximal tumor response. Taxanes have demonstrated excellent activity in breast carcinoma; however, tumor-specific factors that predict clinical response have not been characterized thoroughly. The authors performed a historic review evaluating the association of tumor prognostic factors and response to neoadjuvant cyclophosphamide and doxorubicin (AC) with or without docetaxel (D) (AC vs. AC+D) in 121 women who previously were enrolled in a Phase III, randomized, clinical trial. Using pretreatment biopsy materials, immunohistochemical studies were performed for estrogen receptor (ER), progesterone receptor (PR), HER-2/neu, p53, and Ki-67. Outcome variables were pathologic complete response (pCR) and positive clinical response (cPOS), which was defined as a >/= 50% regression in clinical tumor size prior to surgery. In a multivariate analysis that controlled for tumor size and lymph node status, improved cPOS rates were observed with the addition of docetaxel in women with HER-2/neu-negative tumors (81% vs. 51%; P < 0.05), yielding an adjusted odds ratio of 3.5 (95% confidence interval, 1.2-13.0) in favor of docetaxel. Women who had HER-2/neu-negative tumors appeared to have a lower response rate with AC alone compared with women who had HER-2/neu-positive tumors (51% vs. 75%; P = 0.06), but response rates were matched when docetaxel was added (81% vs. 78%; P = 0.99). ER, PR, p53, and Ki-67 results were not associated significantly with response rates. HER-2/neu status may predict improved clinical response rates from the addition of docetaxel to anthracycline-based neoadjuvant chemotherapy. Docetaxel may "rescue" the response in women who have HER-2/neu-negative tumors to match that observed in women who have HER-2/neu-positive tumors treated with AC alone.

Regulation of cellular glutathione modulates nuclear accumulation of daunorubicin in human MCF7 cells overexpressing multidrug resistance associated protein

Multidrug resistance (MDR) is frequently associated with the overexpression of P-glycoprotein (Pgp) and/or multidrug resistance associated protein (MRP1), both members of the ABC superfamily of transporters. Pgp and MRP1 function as ATP-dependent efflux pumps that extrude cytotoxic drugs from tumour cells. Glutathione (GSH) has been considered to play an important role in the MRP1-mediated MDR. In our study, we examined the effects of buthionine sulphoximine (BSO), an inhibitor of GSH biosynthesis, on the nuclear accumulation of daunorubicin (DNR), in etoposide (VP16) and doxorubicin (ADR) resistant MCF7 cell lines, overexpressing respectively MRP1 (MCF7/VP) and Pgp (MCF7/ADR). The study of DNR transport was carried out using scanning confocal microspectrofluorometry. This technique allows the determination of the nuclear accumulation of anthracyclines in single living tumour cells. Treatment of MCF7/VP cells with BSO increased the sensitivity of these cells to DNR whilst the cytotoxicity of the drug in MCF7/ADR cells remained unchanged. In MCF7 resistant cells treated with BSO, their GSH level decreased as observed by confocal microscopy. DNR nuclear accumulation in MCF7/VP cells was increased by BSO whereas in MCF7/ADR cells BSO was unable to significantly increase the DNR nuclear accumulation. These data suggest a requirement for GSH in MRP1-mediated resistance whilst the nuclear efflux of GSH conjugates is probably not the primary mechanism of Pgp-mediated MDR. Finally, BSO might be a useful agent in clinical assays for facilitating detection of MRP1 expression.

Study on the anticarcinogenic effect and acute toxicity of liver-targeting mitoxantrone nanoparticles.

AIM:To study the anticarcinogenic effect and acute toxicity of liver targeting mitoxantrone nanospheres.METHODS:The anticarcinogenic effect of mitoxan-trone polybutylcyanoacrylate nanoparticles (DHAQ-PBCA-NP) was investigated by using heterotopic and orthotopic transplantation models of human hepatocellular carcinoma (HCC) in nude mice and was compared with mitoxantrone (DHAQ) and doxorubicin (ADR).The acute toxicity of DHAQ-PBCA-NP lyophilized injection in mice was also studied.RESULTS:The tumor inhibition rates of ADR, DHAQ, DHAQ-PBCA-NP to orthotopically transplanted HCC were 60.07%, 67.49% and 99.44%, respectively, but regard to heterotopically transplanted HCC, these were 80.03%, 86.18% and 92.90%, which were concordant with the results acquired by mitosis counting and proliferating cell nuclear antigen (PCNA). After iv administration to mice with DHAQ-PBCA-NP, the LD(50) was 16.9mg/kg ± 3.9mg/kg, no obvious local irritation was observed and there was no significant damage to the structure of liver cells, and that of the heart, spleen and kidneys.CONCLUSION:The effect of DHAQ-PBCA-NP was significantly higher than that of DHAQ and ADR in the anti orthotopically transplanted HCC and the acute toxicity was relatively low.

Transport mechanism of anthracycline derivatives in human leukemia cell lines: uptake and efflux of daunorubicin and doxorubicin in HL60 and its resistant cells and comparison with those of pirarubicin.

We examined the transport mechanisms of daunorubicin (DNR) and doxorubicin (ADR) in HL60 and HL60/THP cells which were the non-P-glycoprotein-mediated resistant clone of the parent HL60 cells and showed a low degree of resistance, and compared them with those of pirarubicin (THP). In both lines, it appeared that the uptakes of DNR and ADR were time-, temperature- and concentration-dependent and energy independent, and the transport of DNR consisted of saturable and nonsaturable components. They were pumped out from the cells time-, temperature- and energy-dependently. There were no differences in the accumulation amount of either DNR or ADR between HL60 and HL60/THP cells. Comparing the transport of DNR or ADR with that of THP, the uptake amounts of DNR and THP were approximately equal, and were greater than that of ADR in both types of cell. In cis-inhibition experiments, DNR inhibited the THP uptake noncompetitively in the parent and resistant cells, in contradiction of the previously reported result in which ADR showed competitive inhibition (Nagasawa, K. et al., Cancer Chemother. Pharmacol., in press). The THP accumulation appeared to be increased by preload of DNR and ADR, indicating a counter transport. Thus, DNR and ADR as well as THP might be incorporated via a common carrier-mediated transport system, but DNR uptake in part appeared to follow a nonsaturable transport, and its binding site in the carrier might differ from that of THP and ADR in both HL60 and HL60/THP cells.

Transport mechanisms of anthracycline derivatives in human leukemia cell lines: uptake of pirarubicin, daunorubicin and doxorubicin by K562 and multidrug-resistant K562/ADM cells.

We studied the uptake mechanisms of anthracycline derivatives, pirarubicin (THP), daunorubicin (DNR) and doxorubicin (ADR), in K562 and multidrug-resistant K562/ADM cells, which overexpress a multidrug efflux pump P-glycoprotein (P-gp). The uptake of THP, DNR and ADR by K562 or K562/ADM cells was time-, temperature- and concentration-dependent. The THP and ADR uptake by the parental cells was not affected by treatment with 4 mM 2,4-dinitrophenol (DNP) alone or DNP plus a P-gp specific inhibitor, cyclosporin A (CyA, 10 microM), while the DNR uptake in the DNP treatment group was significantly greater than that in the control group. There was no difference in the uptake of THP between DNP-pretreated K562 cells and DNP plus CyA-pretreated K562/ADM cells. The uptake of DNR or ADR was almost equal in both types of cell treated with DNP alone. Every kinetic constant for THP, DNR and ADR uptake by the sensitive cells was approximately equal to that in the resistant cells, respectively, under the above conditions. THP uptake was noncompetitively inhibited and stimulated on simultaneous treatment and preloading, respectively, of DNR or ADR in each type of cell. ADR showed noncompetitive inhibition of DNR uptake by either type of cell. Therefore, it was suggested that a common carrier-mediated transport system was involved in the uptake of THP, DNR and ADR, and that their binding sites in the carrier might be different from one another in both K562 and K562/ADM cells.

Transport mechanism of pirarubicin in human mononuclear cells.

We studied the transport mechanism of pirarubicin (THP) in mononuclear cells (MNCs) obtained from healthy human donors. The THP uptake was time-, temperature-, concentration- and energy (in part)-dependent. The uptake of daunorubicin (DNR) and doxorubicin (ADR) was also concentration-dependent, and the transport of ADR consisted of saturable and nonsaturable components. In cis-inhibition experiments, ADR inhibited both THP and DNR uptake noncompetitively, while DNR showed competitive inhibition of the uptake of THP. The THP uptake rate appeared to be increased by preloading DNR, indicating a trans-stimulatory effect, but not with ADR. These results suggest that THP and DNR were taken up into MNCs via a common carrier-mediated transport system, but that the carrier of ADR might differ from that of THP and DNR. Furthermore, apparent differences in the affinity for the carrier, transport efficacy and substrate specificity of the transporter between MNCs and the human leukemia cell lines (HL60 and K562) were indicated.

Initial observations on the effects of delta 9-tetrahydrocannabinol on the plasma pharmacokinetics of cyclophosphamide and doxorubicin.

We studied the effect of THC upon the pharmacokinetics of cyclophosphamide (CTX) and doxorubicin (ADR). Plasma THC was determined by RIA. Plasma concentrations of CTX and ADR were measured by GLC and fluorescence, respectively. RIA confirmed plasma levels of THC greater than 20 ng/ml for patients who received THC. CTX half-life was not significantly changed with use of THC (7.7 +/- 3.6 hours without versus 5.25 +/- 2.6 hours with THC). ADR half-life with THC was greater than without THC (175 +/- 197 hours versus 92 +/- 92 hours, respectively). Total drug exposure as determined by areas under the curves were similar (12.4 +/- 6 microM . hr without versus 13.8 +/- 4 microM . hr with THC). These preliminary data suggest that RIA is reliable for assessing THC plasma concentrations. THC induces no apparent alterations of CTX or ADR pharmacokinetics.