Determining the cytotoxicity of methotrexate‐loaded microemulsion on human breast,ovarian, and prostate carcinoma cell lines: a new modality for an old drug

The main objective of this study was to develop an optimal methotrexate (MTX) microemulsion (M‐MTX) and to evaluate the suppressive effect of MTX‐loaded microemulsion on human breast, ovarian, and prostate carcinoma cell lines. The microemulsion was made up of corn‐oil as the oil phase, a mixture of Cremophore EL and Span 80 as surfactants, and isopropyl alcohol as co‐surfactant and 0.1 N NaOH as the aqueous phase. MTX (0.1 mg/mL MTX ) was added into the microemulsion at the last stage. The pH of M‐MTX was adjusted to pH 8±0.1 and the physicochemical stability of the formulation was observed. The particle size distribution was measured by Zetasizer 3000 HSA. The mean droplet diameters of M‐MTX were determined as 13.3±0.1 nm (poly dispersity index=530±0.01). The antitumor effects of M‐MTX were examined on human breast (MCF‐7), ovarian (OVCAR), and prostate (DU 145) carcinoma cell lines. It was clearly demonstrated that M‐MTX had a significant cytotoxic effect on different carcinoma cell lines and the cytotoxic effect of M‐MTX was significantly more than that of commercial preparation (C‐MTX) (P<0.05). According to the in vitro cytotoxicity studies, it can be concluded that when MTX was incorporated into the microemulsion (M‐MTX), which is a new drug carrier system, it suppresses tumour cell growth on multiple tumor lines. These results indicate that M‐MTX may be effective as an antitumor agent that induces apoptosis. Drug Dev Res 2009. © 2009 Wiley‐Liss, Inc.     

Preparation of arsenic trioxide-loaded microemulsion and its enhanced cytotoxicity on MCF-7 breast carcinoma cell line

In this study, an injectable microemulsion of arsenic trioxide (As2O3-M) was prepared for intratumoral injection and the suppressive effect of As2O3-loaded microemulsion on human breast cancer cells MCF-7 was compared with those of a solution of the drug. Microemulsion was made up of soybean oil as oil phase, a mixture of Brij 58 and Span 80 as surfactants, absolute ethanol as cosurfactant, and bidistilled water containing As2O3 solution as the aqueous phase. Microemulsion formulation contains 5 x 10(-6) M As2O3. The pH of As2O3-M was adjusted to 7.35 +/- 0.1 and the physicochemical stability of the formulation was observed. The particle size distribution and zeta potential of As2O3-M were measured by Zetasizer 3000 HSA. The mean droplet diameters of As2O3-M were determined as 8.6 +/- 0.4 nm. As2O3-M exhibited 13.1 +/- 0.9 mV zeta potential. The formulation was physically stable for 12 months at room temperature when kept in ampule forms, as well as after autoclaving at 110 degrees C for 30 min. The antitumor effects of As2O3-M were examined on human breast cancer cells MCF-7. It was clearly demonstrated that As2O3-M had a significant cytotoxic effect on breast cancer cell lines, and the cytotoxic effect of As2O3-M was significantly more than that of regular As2O3 solutions. Even approximately 3000 times diluted microemulsion formulation loaded with 5 x 10(-6) M As2O3 showed a cytotoxic effect. As a result, this diluted concentration (approximately 1.6 x 10(-9) M) was found 1000 times more effective than regular As2O3 solutions (5 x 10(-6) M). According to the in vitro cytotoxicity studies, we concluded that when As2O3 was incorporated into the microemulsion (As2O3-M), which is a new drug carrier system, it suppresses tumor cell growth on multiple tumor lines. These results indicate that As2O3-M may exert a low cytotoxic effect on normal cells and may be effective as an antitumor agent that induces apoptosis.     

短肽修饰的甲氨蝶呤的制备及细胞毒性作用

目的:制备促黄体激素释放激素-甲氨蝶呤(LH-RH-MTX)并考察其对表达有促黄体激素释放激素(LH-RH)受体的肿瘤细胞的毒性作用。方法:以临床抗恶性肿瘤药物甲氨蝶呤(MTX)为模型药物,以促黄体激素释放激素[D-Lys6]LH-RH为导向物,通过共价键连接制备LH-RH-MTX。应用MTT法分别考察游离MTX和LH-RH-MTX对鼻咽癌HNE-1及前列腺癌PC-3细胞株的细胞毒性作用。结果:MTT法结果表明游离MTX和LH-RH-MTX对2种细胞株均有生长抑制作用,对前列腺癌PC-3细胞的抑制率大于鼻咽癌HNE-1细胞;给药12h后抑制率达到最大;当药物质量浓度为200.0μg.L-1,作用12h后:LH-RH-MTX对前列腺癌PC-3细胞的抑制率可高达97.73%,游离MTX为49.90%;LH-RH-MTX对鼻咽癌HNE-1细胞的抑制率为38.92%,游离MTX为7.41%;在同一种细胞株上,LH-RH-MTX的IC50值小于游离MTX。结论:经过短肽修饰的化合物LH-RH-MTX可与表达有LH-RH受体的肿瘤细胞特异性结合并发挥抑制作用,抗肿瘤活性高于游离MTX。提示用LH-RH作为TDDS的导向物...     

The novel oral imatinib microemulsions: physical properties,cytotoxicity activities and improved Caco-2 cell permeability

The objective of this study was to formulate imatinib (IM) loaded to water-in-oil (w/o) microemulsions as an alternative formulation for cancer therapy and to evaluate the cytotoxic effect of microemulsions Caco-2 and MCF-7. Moreover, permeability studies were also performed with Caco-2 cells. W/o microemulsion systems were developed by using pseudo-ternary phase diagram. According to cytotoxicity studies, all formulations did not exert a cytotoxic effect on Caco-2 cells. Furthermore, all formulations had a significant cytotoxic effect on MCF-7 cells and the cytotoxic effect of M3IM was significantly more than that of other microemulsions and IM solution (p?<?0.05). The permeability studies of IM across Caco-2 cells showed that permeability value from apical to basolateral was higher than permeability value of other formulations. In conclusion, the microemulsion formulations as a drug carrier, especially M3IM formulation, may be used as an effective alternative breast cancer therapy for oral delivery of IM.     

Differential subcellular distribution of mitoxantrone in relation to chemosensitization in two human breast cancer cell lines.

The present work investigates the relationship between cancer cell chemosensitivity and subcellular distribution, molecular interaction, and metabolism of an anticancer drug. To get insights into this relationship, we took advantage of the differential sensitivity of two breast cancer cell lines, MDA-MB-231 and MCF-7, to anthracyclines, along with the property of docosahexaenoic acid (DHA, 22:6n-3), to differentially enhance their cytotoxic activity. The fluorescent drug mitoxantrone (MTX) was used because of the possibility to study its subcellular accumulation by confocal spectral imaging (CSI). The use of CSI allowed us to obtain semiquantitative maps of four intracellular species: nuclear MTX bound to DNA, MTX oxidative metabolite in endoplasmic reticulum, cytosolic MTX, and finally, MTX in a low polarity environment characteristic of membranes. MDA-MB-231 cells were found to be more sensitive to MTX (IC50 = 18 nM) than MCF-7 cells (IC50 = 196 nM). According to fluorescence levels, the nuclear and cytosolic MTX content was higher in MCF-7 than in MDA-MB-231 cells, indicating that mechanisms other than nuclear MTX accumulation account for chemosensitivity. In the cytosol, the relative proportion of oxidized MTX was higher in MDA-MB-231 (60%) than in MCF-7 (7%) cells. DHA sensitized MDA-MB-231 (approximately 4-fold) but not MCF-7 cells to MTX and increased MTX accumulation by 1.5-fold in MDA-MB-231 cells only. The DHA-stimulated accumulation of MTX was attributed mainly to the oxidative metabolite. Antioxidant N-acetyl-L-cysteine inhibited the DHA effect on both metabolite accumulation and cell sensitization to MTX. We conclude that drug metabolism and compartmentalization are associated with cell chemosensitization, and the related cytotoxicity mechanisms may involve oxidative stress.     

Methotrexate conjugated to gold nanoparticles inhibits tumor growth in a syngeneic lung tumor model

Methotrexate (MTX), a stoichiometric inhibitor of dihydrofolate reductase, is a chemotherapeutic agent for treating a variety of neoplasms. Impairment of drug import into cells and increase in drug export from cells may render cells resistant to MTX. MTX, when locally administered in a soluble form, is rapidly absorbed through capillaries into the circulatory system, which may also account for therapeutic failure in patients. To retain MTX within tumor cells for longer duration and alter its pharmacokinetic behavior, we proposed a new formulation of MTX bound to the gold nanoparticle (AuNP) that serves as drug carriers. In this study, we developed the MTX-AuNP conjugate and examined its cytotoxic effect in vitro and antitumor effect in vivo. Spectroscopic examinations revealed that MTX can be directly bound onto AuNP via the carboxyl group (-COOH) to form the MTX-AuNP complex and kinetically released from the nanoparticles. The accumulation of MTX is faster and higher in tumor cells treated with MTX-AuNP than that treated with free MTX. Notably, MTX-AuNP shows higher cytotoxic effects on several tumor cell lines compared with an equal dose of free MTX. This can be attributed to the "concentrated effect" of MTX-AuNP. Administration of MTX-AuNP suppresses tumor growth in a mouse ascites model of Lewis lung carcinoma (LL2), whereas an equal dose of free MTX had no antitumor effect. In conclusion, these results suggest that by combining nanomaterials with anticancer drugs MTX-AuNP may be more effective than free MTX for cancer treatment.     

Diazene JK-279: potential anticancer drug

The aim of this study was to examine the cytotoxic effect of 10 newly synthesized diazenecarboxamides (diazenes). Using a modified colorimetric MTT assay, their cytotoxicity was determined on 10 human cell lines: cervical carcinoma parental and cisplatin-resistant cells, laryngeal carcinoma parental and cisplatin- and vincristine-resistant cells, glioblastoma parental and cisplatin-resistant cells, breast adenocarcinoma parental and doxorubicin-resistant cells, and mammary carcinoma cells. Results show that diazene JK-279 was most effective, reducing significantly the cell survival of all 10 cell lines examined, including five drug-resistant cell lines. A cytotoxic effect was observed also on nine from 10 cell lines for diazene JK-835. A small reduction in cell survival was obtained (mainly for highest drug concentrations) for diazenes LV-57 and MG-19 on two cell lines, and JK-429 and JK-913 on one cell line. Other diazenes did not demonstrate any cytotoxic activity. The results encourage further research on diazene JK-279 as a potential anticancer drug.     

In vitro characterization and growth inhibition effect of nanostructured lipid carriers for controlled delivery of methotrexate

The present study describes the design and characterization of nanostructured lipid carriers (NLCs) for controlled delivery of methotrexate (MTX). A series of NLCs with or without MTX were prepared using different ratios of liquid–lipid to solid–lipid and type and concentration of surfactants. The effect of different formulation parameters on the physical properties of NLCs, entrapment efficiency of MTX and in vitro drug release was evaluated. In addition, the in vitro delivery and cytotoxicity of MTX-loaded NLCs against human prostate cancer DU-145 cells and ovarian human cancer A2780 cells were investigated. Drug loading capacity, particle size and surface charge of the prepared NLCs and the in vitro MTX release were affected by the formulation parameters. In vitro growth inhibition assay using DU-145 and A2780 cancer cell lines showed that drug-free NLCs maintained cell viability while MTX-loaded NLCs inhibited the growth of both cell lines. In addition, MTX-loaded NLCs showed superior inhibitory effect on cell growth over the free drug especially in A2780 cell lines and a higher cytotoxic effect on DU-145 at higher drug concentration. The results of the current study warrant further exploration for the use NLCs as a controlled delivery system for chemotherapeutic agents.     

Preparation of Arsenic Trioxide-Loaded Microemulsion and Its Enhanced Cytotoxicity on MCF-7 Breast Carcinoma Cell Line

In this study, an injectable microemulsion of arsenic trioxide (As₂O₃-M) was prepared for intratumoral injection and the suppressive effect of As₂O₃-loaded microemulsion on human breast cancer cells MCF-7 was compared with those of a solution of the drug. Microemulsion was made up of soybean oil as oil phase, a mixture of Brij 58 and Span 80 as surfactants, absolute ethanol as cosurfactant, and bidistilled water containing As₂O₃ solution as the aqueous phase. Microemulsion formulation contains 5 × 10^?6 M As₂O₃. The pH of As₂O₃-M was adjusted to 7.35 ± 0.1 and the physicochemical stability of the formulation was observed. The particle size distribution and zeta potential of As₂O₃-M were measured by Zetasizer 3000 HSA. The mean droplet diameters of As₂O₃-M were determined as 8.6 ± 0.4 nm. As₂O₃-M exhibited 13.1 ± 0.9 mV zeta potential. The formulation was physically stable for 12 months at room temperature when kept in ampule forms, as well as after autoclaving at 110°C for 30 min. The antitumor effects of As₂O₃-M were examined on human breast cancer cells MCF-7. It was clearly demonstrated that As₂O₃-M had a significant cytotoxic effect on breast cancer cell lines, and the cytotoxic effect of As₂O₃-M was significantly more than that of regular As₂O₃ solutions. Even ～ 3000 times diluted microemulsion formulation loaded with 5 × 10^?6 M As₂O₃ showed a cytotoxic effect. As a result, this diluted concentration (～1.6 × 10^?9 M) was found 1000 times more effective than regular As₂O₃ solutions (5 × 10^?6 M). According to the in vitro cytotoxicity studies, we concluded that when As₂O₃ was incorporated into the microemulsion (As₂O₃-M), which is a new drug carrier system, it suppresses tumor cell growth on multiple tumor lines. These results indicate that As₂O₃-M may exert a low cytotoxic effect on normal cells and may be effective as an antitumor agent that induces apoptosis.     

Establishment and characterization of new murine breast cancer cell lines

The establishment of two new breast cancer cell lines, MXT+ and MXT-, derived from the murine breast cancer models MXT-M-3,2 MC (hormone-sensitive) and MXT-M-3,2 (ovex) MC (hormone-insensitive), is described. Characterization of the cell lines was performed by investigation of morphology, steroid hormone receptor state, growth kinetics, and drug response as well as by cytogenetic analysis. MXT+ contains estrogen receptors (ER; 6.9 fmol/mg protein) as well as progesterone receptors (PgR; 9.2 fmol/mg protein) and therefore is inhibited by tamoxifen (Tam). MXT- proved to be ER- but PgR+ (23.4 fmol/mg protein) and, as expected, resistant against Tam. The sensitivity of MXT+ and MXT- against a pattern of therapeutically established anti-breast cancer drugs (cDDP, cisplatin; JM-8, carboplatin; DX, adriamycin; 5-FU, 5-fluorouracil; MTX, methotrexate; VLB vinblastine) was studied by use of a computerized, kinetic chemosensitivity assay based on quantification of biomass by staining cells with crystal violet. For each compound the inhibition profile reflecting cytostatic, transient cytotoxic, or cytocidal drug effects as well as development of resistance was evaluated. The following order of activity was found: MTX > VLB > or = DX > cDDP > or = 5-FU > JM-8. The test data of 5-FU, VLB, cDDP, and Tam on MXT+ as well as on MXT- were compared with those from studies on ER+ and ER- human breast cancer cell lines (MCF-7, ZR-75-1, T-47-D, and MDA-MB-231, respectively). They revealed comparable inhibition profiles and sensitivities of human and murine breast cancer cell lines, an indication that the results achieved in combined in vitro-/in vivo tests by use of the murine test models MXT+, MXT-, MXT-M-3,2 MC, and MXT-M-3,2(ovex) MC are relevant for therapy in humans.     

The kinetics of methotrexate polyglutamate formation and efflux in a human breast cancer cell line (MDA.MB.436): the effect of insulin

The rate and extent of the formation of methotrexate poly-γ-glutamates has been studied in a human breast cancer cell line (MDA.MB.436). Cells were exposed to medium containing 10^?7 M radiolabelled methotrexate (MTX) for various periods. Cell extracts were subjected to gel filtration on Bio-Gel P2 and radioactivity in each fraction determined. This has allowed quantitation of the relative amounts of MTX and its polyglutamate derivatives. It has been proposed that MTX polyglutamates may act as storage forms of the drug which could result in prolonged cytotoxicity. This possibility was investigated by determining the ability of the cell line to retain MTX and its olyglutamate derivatives for periods of up to 48 hr after removal of MTX from the incubation medium. Our results show that the lower mol. wt polyglutamates (< three extra glutamic acid residues) rapidly efflux from the cell, while the higher mol. wt species (> three glutamic acid residues) are extensively retained, having an efflux half-life of approximately 30 hr. It has been shown that insulin may potentiate the cytotoxicity of MTX to breast cancer cells in vitro. Our results indicate that in the MDA.MB.436 cell line the total intracellular drug at the steady state is unaffected by insulin (10^?6 M). However insulin does result in a 30% increase in the contribution of the higher mol. wt polyglutamates to the total intracellular drug. Our data therefore suggest that the ability of insulin to potentiate the cytotoxic effects of MTX may be related to the hormone's ability to modulate the synthesis of MTX polyglutamates.     

Lack of an effect of breast cancer resistance protein (BCRP/ABCG2) overexpression on methotrexate polyglutamate export and folate accumulation in a human breast cancer cell line

Accumulation of methotrexate (MTX) and its polyglutamates (PGs) has been recognized as an important factor in MTX efficacy. We have previously described a multidrug-resistant human breast cancer cell line, MCF7/MX, that exhibits reduced accumulation of total MTX as well as MTX-PGs, and that is resistant to continuous MTX exposure [Volk EL, Rohde K, Rhee M, McGuire JJ, Doyle LA, Ross DD, et al. Methotrexate cross-resistance in a mitoxantrone selected multidrug-resistant MCF7 breast cancer cell line is due to enhanced energy-dependent drug efflux. Cancer Res 2000;60:3514-21]. These cells express high levels of the breast cancer resistance protein (BCRP/ABCG2) that has been shown to actively transport MTX and short-chain MTX-PGs in vitro. However, the effect of BCRP on MTX-PG accumulation in intact cells was unclear. Here, we show that MTX transport by BCRP is required for the observed lower levels of MTX-PGs in the resistant cells. When BCRP was inhibited with fumitremorgin C, or in cells expressing a mutated form of BCRP that is unable to transport MTX, MTX-PG accumulation was similar or even higher than that in the parental cells that do not express BCRP. Concomitantly, there was increased inhibition of thymidylate synthase. It had previously been suggested that BCRP-mediated efflux of MTX-PGs contributed to the reduced MTX-PG accumulation. However, we found no evidence of BCRP-mediated efflux of MTX-PGs from intact cells, suggesting that direct efflux of MTX-PGs does not play a major role in MTX resistance. Together, these data show that BCRP overexpression can cause a reduction in total MTX accumulation as well as a reduction in the proportion of long-chain MTX-PGs. In contrast, BCRP overexpression did not affect natural folate accumulation or the relative distribution of folylpolyglutamates in the resistant, as compared to the parental, cells. Thus, it appears that BCRP overexpression affects the metabolism of the antifolate MTX, but not that of natural folates, although indirect effects cannot be excluded.     

Enhancement of methotrexate cytotoxicity towards the MDA.MB.436 human breast cancer cell line by dipyridamole. The role of methotrexate polyglutamates

MTX and dipyridamole are synergistic in their toxicity towards the MDA.MB.436 human breast cancer cell line. Dipyridamole increases net MTX uptake into the cells and increases the intracellular levels of MTXG7 to G10, the highest molecular weight polyglutamyl derivatives of MTX detected. During a recovery period, after completion of exposure to MTX with and without dipyridamole, levels of MTXG7 to G10 remained elevated in dipyridamole treated cells by comparison with controls. Dipyridamole, which has no intrinsic effect on cell growth, transforms a cytostatic response of MDA.MB.436 cells towards MTX into a cytotoxic response. The effect of dipyridamole is not mediated through an increase in prostacyclin biosynthesis.     

Transdermal delivery of methotrexate: iontophoretic delivery from hydrogels and passive delivery from microemulsions

In vitro assays were performed to investigate the effectiveness of transdermal administration of methotrexate (MTX) by iontophoretic delivery from two types of hydrogel and passive delivery from two types of microemulsion. Both iontophoretic delivery of MTX from hydrogels and passive delivery from microemulsions were more effective than passive delivery from aqueous solutions of the drug. In the iontophoretic delivery assays, the type of hydrogel used and the concentration of the drug in the loading solution had little influence on effectiveness of delivery. In the passive delivery assays, we used both water/oil (w/o) and oil/water (o/w) microemulsions: effectiveness of delivery was higher from o/w systems. At the end of all assays, significant amounts of MTX were detected in the skin. These results suggest that both hydrogels and microemulsions may be of value for the topical administration of MTX in the treatment of psoriasis.     

Discrimination among reduced folates and methotrexate as transport substrates by a phenylalanine substitution for serine within the predicted eighth transmembrane domain of the reduced folate carrier.

A phenylalanine substitution for serine in the reduced folate carrier at residue 309 (RFC1-S309F) was identified in a methotrexate (MTX)-resistant cell line selected with 5-formyltetrahydrofolate (5-CHO-THF) as the sole folate source. The transport characteristics of the mutated carrier were studied by transfection into the MTXrA line, which lacks endogenous RFC1 function. The level of expression of carrier in the cell lines studied was determined by specific surface binding of 5-methyltetrahydrofolate (5-CH3-THF). Influx of 5-CH3-THF and 5-CHO-THF mediated by RFC1-S309F was 20- and 7-fold greater than that of MTX, respectively. Consistent with the influx difference between 5-CHO-THF and MTX, the growth requirement (EC50) for 5-CHO-THF in MTXrA-S309F cells was decreased by a factor of 9, while the MTX IC50 was reduced by a factor of only approximately 2 as compared with the recipient MTXrA cells. The decrease in 5-CH3-THF influx mediated by the mutated carrier was attributed to a decrease in the mobility of the 5-CH3-THF-carrier complex, since the influx Kt was essentially unchanged. However, the reduction in 5-CHO-THF and MTX influx was attributed to decreases in both carrier affinity and Vmax, although the decline in the MTX influx Vmax appeared to be much greater than for 5-CHO-THF. The inhibitory effect of chloride on 5-CHO-THF influx observed for L1210 cells was eliminated in the MTXrA-S309F line. This study represents another example of a single mutation in RFC1 that markedly impairs MTX influx but partially preserves transport of reduced folates when cells are selected with 5-CHO-THF as the available folate substrate. The data indicate that residues in the predicted eighth transmembrane domain of RFC1 can play an important role in the selectivity of folate binding and the mobility of the carrier-substrate complex.     

Preparation and characterization of a biodegradable drug targeting system for anticancer drug delivery: Microsphere-antibody conjugate

Targeted delivery of anticancer drugs is one of the most actively pursued goals in anticancer chemotherapy. A major disadvantage of anticancer drugs is their lack of selectivity for tumour tissue, which causes severe side effects and results in low cure rates. Any strategy by which a cytotoxic drug is targeted to the tumour, thus increasing the therapeutic index of the drug, is a way of improving cancer chemotherapy and minimizing systematic toxicity. This study covers the preparation of the gelatin microsphere (GM)-anti-bovine serum albumin (anti-BSA) conjugate for the development of a drug targeting approach for anticancer drug delivery. Microspheres of 5% (w/v) gelatin content were prepared by crosslinking with glutaraldehyde (GTA) at 0.05 and 0.50% (v/v) concentration. Microspheres were in the size range of 71–141 μm. The suitability of these microspheres as drug carriers for anticancer drug delivery was investigated in vitro by studying the release profiles of loaded methotrexate (MTX) and 5-fluorouracil (5-FU) and the cytotoxicities on cancer cell lines. The in vitro MTX release profiles (～22–46% released in 24 h depending on the amount of GTA used) were much slower compared to 5-FU (～42–91% released in 24 h). Both drugs demonstrated an initial fast release, which was followed by gradual, sustained drug release. The MTT cytotoxicity test results of GMs loaded with 5-FU and MTX showed ～54–70% and ～52–67% cytotoxicities in 4 days. In general, incorporation of MTX and 5-FU in microspheres enhanced the cytotoxic effect in a more prolonged manner compared to the free drugs. Gelatin micospheres were chemically conjugated to anti-BSA and the antigen–antibody activities were studied by immunofluorescence. Results indicated ～80% binding with conjugated anti-BSA and BSA-FITC. Based on their low cytotoxicity and the high antigen binding efficiencies, anti-BSA conjugated gelatin microspheres could be suitable targeted drug carrier systems for selective and long-term delivery of anticancer drugs to a specific body compartment (i.e. bladder cancer).     

Undecylprodigiosin selectively induces apoptosis in human breast carcinoma cells independent of p53

Undecylprodigiosin (UP) is a bacterial bioactive metabolite produced by Streptomyces and Serratia. In this study, we explored the anticancer effect of UP. Human breast carcinoma cell lines BT-20, MCF-7, MDA-MB-231 and T47D and one nonmalignant human breast epithelial cell line, MCF-10A, were tested in this study. We found that UP exerted a potent cytotoxicity against all breast carcinoma cell lines in a dose- and time-dependent manner. In contrast, UP showed limited toxicity to MCF-10A cells, indicating UP's cytotoxic effect is selective for malignant cells. UP's cytotoxic effect was due to apoptosis, as confirmed by positive TUNEL signals, annexin V-binding, caspase 9 activation and PARP cleavage. Notably, UP-induced apoptosis was blocked by the pan-caspase inhibitor z-VAD.fmk, further indicating the involvement of caspase activity. Moreover, UP caused a marked decrease of the levels of antiapoptotic BCL-X(L), Survivin and XIAP while enhancing the levels of proapoptotic BIK, BIM, MCL-1S and NOXA, consequently favoring induction of apoptosis. Additionally, we found that cells with functional p53 (MCF-7, T47D) or mutant p53 (BT-20, MDA-MB-231) were both susceptible to UP's cytotoxicity. Importantly, UP was able to induce apoptosis in MCF-7 cells with p53 knockdown by RNA interference, confirming the dispensability of p53 in UP-induced apoptosis. Overall, our results establish that UP induces p53-independent apoptosis in breast carcinoma cells with no marked toxicity to nonmalignant cells, raising the possibility of its use as a new chemotherapeutic drug for breast cancer irrespective of p53 status.     

穿龙骨刺片联合艾瑞昔布治疗膝骨关节炎的疗效及对氧化应激的影响

目的探讨穿龙骨刺胶囊联合艾瑞昔布治疗膝骨关节炎的临床效果。方法选取2020年1月—2021年1月在郑州大学第一附属医院治疗的102例膝骨关节炎患者,根据用药差别分为对照组和治疗组,每组各51例。对照组口服艾瑞昔布片,0.1 g/次,2次/d;治疗组在对照组基础上口服穿龙骨刺片,4 g/次,3次/d。两组均经8周治疗。观察两组的临床疗效,比较两组临床症状改善时间、相关评分和血清氧化应激指标。结果经治疗,治疗组总有效率是98.04%,显著高于对照组的82.35%(P0.05)。经治疗,治疗组膝关节疼痛、肿胀、活动受限改善时间明显短于对照组(P0.05)。经治疗,两组VAS评分、WOMAC评分、ISOA评分均较治疗前显著降低,但Lysholm评分显著升高(P0.05);且治疗后治疗组相关评分改善优于对照组(P0.05)。经治疗,两组患者血清超氧化物歧化酶(SOD)显著升高,但丙二醛(MDA)水平显著降低(P0.05);治疗后,治疗组血清氧化应激水平改善优于对照组(P0.05)。结论穿龙骨刺胶囊联合艾瑞昔布治疗膝骨关节炎具有较好的临床疗效,可有效改善患者临床症状,改善关节功能,抑制氧化应激反应,有着良好的临床应用价值。