Cellular stress induced by photodynamic reaction with CoTPPS and MnTMPyPCl5 in combination with electroporation in human colon adenocarcinoma cell lines (LoVo and LoVoDX)

Two porphyrins, CoTPPS and MnTMPyPCl₅, were tested for their photodynamic activity and potential novel use in a therapy of human cancers. We investigated an effect of photodynamic reaction (PDR), electroporation (EP) and their combination (electro-photodynamic reaction [EP-PDR]) on human colon adenocarcinoma cell lines (LoVo and resistant to doxorubicin LoVoDX), human breast adenocarcinoma (wild type MCF-7/WT and resistant to doxorubicin MCF-7/DOX), and human melanoma (Me45). The efficiency of macromolecules transport was examined with cytofluorymetry by assessing the degree of propidium iodide (PI) penetration. Additionally, cellular ultrastructure after EP was evaluated. We determined cyto- and photo-cytotoxic effect on the cells viability (MTT assay) after standard PDR and PDR combined with EP. Intracellular distribution and mitochondrial colocalization of both porphyrins was also performed. The experiments proved that both complexes exhibit desirable photodynamic properties on LoVo LoVoDX cells, and EP effectively supports photodynamic method in this type of cancer. The application of EP provided shorter time of incubation (only 10 min) and enhanced effect of applied therapy. The porphyrins did not affect the MCF-7 and Me45 cell lines.     

Modulating ALA-PDT efficacy of mutlidrug resistant MCF-7 breast cancer cells using ALA prodrug

Multi-drug resistance of breast cancer is a major obstacle in chemotherapy of cancer treatments. Recently it was suggested that photodynamic therapy (PDT) can overcome drug resistance of tumors. ALA-PDT is based on the administration of 5-aminolevulinic acid (ALA), the natural precursor for the PpIX biosynthesis, which is a potent natural photosensitizer. In the present study we used the AlaAcBu, a multifunctional ALA-prodrug for photodynamic inactivation of drug resistant MCF-7/DOX breast cancer cells. Supplementation of low doses (0.2mM) of AlaAcBu to the cells significantly increased accumulation of PpIX in both MCF-7/WT and MCF-7/DOX cells in comparison to ALA, or ALA + butyric acid (BA). In addition, our results show that MCF-7/DOX cells are capable of producing higher levels of porphyrins than MCF-7/WT cells due to low expression of the enzyme ferrochelatase, which inserts iron into the tetra-pyrrol ring to form the end product heme. Light irradiation of the AlaAcBu treated cells activated efficient photodynamic killing of MCF-7/DOX cells similar to the parent MCF-7/WT cells, depicted by low mitochondrial enzymatic activity, LDH leakage and decreased cell survival following PDT. These results indicate that the pro-drug AlaAcBu is an effective ALA derivative for PDT treatments of multidrug resistant tumors.     

Effect of curcumin-nanoemulsion associated with photodynamic therapy in breast adenocarcinoma cell line

Curcumin, a natural compound has several antineoplastic activities and is a promising natural photosensitizer used in photodynamic therapy. However, its low solubility in physiological medium limit the clinical use of curcumin. This study aimed to analyze the action of curcumin-nanoemulsion, a new and well-designed Drug Delivery System (DDS+) molecule, used as a photosensitizing agent in photodynamic therapy in an in vitro breast cancer model, MCF-7 cells. The empty nanoemulsion fulfils all necessary requirements to be an excellent DDS. Furthermore, the use of curcumin-nanoemulsion in photodynamic therapy resulted in a high phototoxic effect after activation at 440?nm, decreasing to <10% viable tumor cells after two irradiations and increasing the reactive oxygen species (ROS) production. The use of curcumin-nanoemulsion associated with photodynamic therapy resulted in an increase in the levels of caspase 3/7 activity for the studied MCF-7 cell model, indicating that this therapy triggers a cascade of events that lead to cell death, such as cellular apoptosis. In conclusion, curcumin-nanoemulsion proved to be efficient as a photosensitizing agent, had phototoxic effects, significantly decreased the proliferation of MCF-7 cells and stimulating the ROS production in combination with photodynamic therapy, so, this formulation has a great potential for use in treatment of breast cancer.     

Reversal of doxorubicin resistance by guggulsterone of Commiphora mukul in vivo

Our previous study has shown co-administration of guggulsterone resulted in significant increase in chemosensitivity of multidrug-resistant human breast cancer MCF-7/DOX cells to doxorubicin (DOX) in vitro. The present study was designed to investigate whether guggulsterone had the similar modulatory activities in vivo. MCF-7/DOX and MCF-7 xenograft mice models were established. At the end of the experiment (day 28), doxorubicin treatment alone did not significantly inhibit tumor growth in MCF-7/DOX xenograft, indicating that it retained doxorubicin resistance. Whereas, doxorubicin treatment alone significantly inhibited tumor growth in MCF-7 xenograft, suggesting that it maintained doxorubicin sensitivity. When doxorubicin and guggulsterone were co-administrated, their antitumor activities were augmented in MCF-7/DOX xenograft. However, combination therapy did not enhance the antitumor effects of doxorubicin in MCF-7 xenograft. The expression of proliferative cell nuclear antigens PCNA and Ki67 after doxorubicin treatment alone was not significantly different from that of vehicle group in MCF-7/DOX xenograft. On the contrary, doxorubicin treatment alone significantly reduced PCNA and Ki67 expression in MCF-7 xenograft. Combination therapy also significantly reduced PCNA and Ki67 expression in MCF-7/DOX xenograft, compared to doxorubicin treatment alone. However, combination therapy did not enhance the inhibitory effects of doxorubicin on PCNA and Ki67 expression in MCF-7 xenograft. Examining the apoptotic index by TUNEL assay showed similar results. Further studies demonstrated the inhibitory effects of guggulsterone on Bcl-2 and P-glycoprotein expression were the possible reason to increase chemosensitivity of MCF-7/DOX cells to doxorubicin in vivo. Examining body weight, hematological parameters, hepatic, cardiac and gastrointestinal tracts histopathology revealed that no significant signs of toxicity were related to guggulsterone. Guggulsterone might reverse doxorubicin resistance in vivo, with no severe side effects.     

Interaction of cyanine dyes with nucleic acids: XXXI. Using of polymethine cyanine dyes for the visualization of DNA in agarose gels

Fifteen polymethine cyanine dyes were studied as fluorescent stains for DNA in electrophoretic gels. Among studied cyanines, two dyes CPent V and CCyan 2-O most effectively visualized covalently closed and linear double-stranded DNA molecules in gels under standard conditions using UV-illumination, green filter and black-and-white photo film. Ethidium bromide was 1.2-1.6 times more effective as compared to cyanine dyes in staining of DNA in the concentration range of 8-18 ng, while studied cyanines were more sensitive to DNA quantity above 50 ng.     

Modulation of DNA methylation levels sensitizes doxorubicin-resistant breast adenocarcinoma cells to radiation-induced apoptosis

Chemoresistant tumors often fail to respond to other cytotoxic treatments such as radiation therapy. The mechanisms of chemo- and radiotherapy cross resistance are not fully understood and are believed to be epigenetic in nature. We hypothesize that MCF-7 cells and their doxorubicin-resistant variant MCF-7/DOX cells may exhibit different responses to ionizing radiation due to their dissimilar epigenetic status.Similar to previous studies, we found that MCF-7/DOX cells harbor much lower levels of global DNA methylation than MCF-7 cells. Furthermore, we found that MCF-7/DOX cells had lower background apoptosis levels and were less responsive to radiation than MCF-7 cells. Decreased radiation responsiveness correlated to significant global DNA hypomethylation in MCF-7/DOX cells.Here, for the first time, we show that the radiation resistance of MCF-7/DOX cells can be reversed by an epigenetic treatment - the application of methyl-donor SAM. SAM-mediated reversal of DNA methylation led to elevated radiation sensitivity in MCF-7/DOX cells. Contrarily, application of SAM on the radiation sensitive and higher methylated MCF-7 cells resulted in a decrease in their radiation responsiveness. This data suggests that a fine balance of DNA methylation is needed to insure proper radiation and drug responsiveness.     

Specific fluorescent tracers. Imaging and applications for photodynamic therapy

Our main objective is to enlarge the fluorescence use in biosciences, with especially the photodynamic therapy (PDT) used for cancer treatment as one of the target applications. Meta-tetra(hydroxyphenyl)chlorin (m-THPC) is a second-generation photosensitiser, applied in photodynamic therapy. The localisation of this sensitiser as well as its induced cell death mechanisms in human breast cancer cells (MCF-7 and its resistant subline MCF-7DXR, DXR: doxorubicin) were evaluated using fluorescence microscopy. In addition, we will present two additional routes, whose aims are to create new features to respond to the PDT questioning: firstly, the synthesis of fluorescent tracers, with a particular attention to the presence of hydrophilic groups (glucosamine ring) on the basic fluorophore structure to orientate the localisation of the probe and, secondly, the use of scanning near-field optical microscopy to reach a better resolution for the fluorescence microscopy analysis.     

Association of increased basement membrane invasiveness with absence of estrogen receptor and expression of vimentin in human breast cancer cell lines.

Lack of estrogen receptor (ER) and presence of vimentin (VIM) associate with poor prognosis in human breast cancer. We have explored the relationships between ER, VIM, and invasiveness in human breast cancer cell lines. In the matrigel outgrowth assay, ER+/VIM- (MCF-7, T47D, ZR-75-1), and ER-/VIM- (MDA-MB-468, SK-Br-3) cell lines were uninvasive, while ER-/VIM+ (BT549, MDA-MB-231, MDA-MB-435, MDA-MB-436, Hs578T) lines formed invasive, penetrating colonies. Similarly, ER-/VIM+ cell lines were significantly more invasive than either the ER+/VIM- or ER-/VIM- cell lines in the Boyden chamber chemoinvasion assay. Invasive activity in nude mice was only seen with ER-/VIM+ cell lines MDA-MB-231, MDA-MB-435 and MDA-MB-436. Hs578T cells (ER-/VIM+) showed hematogenous dissemination to the lungs in one of five mice, but lacked local invasion. The ER-/VIM+ MCF-7ADR subline was significantly more active than the MCF-7 cells in vitro, but resembled the wild-type MCF-7 parent in in vivo activity. Data from these cell lines suggest that human breast cancer progression results first in the loss of ER, and subsequently in VIM acquisition, the latter being associated with increased metastatic potential through enhanced invasiveness. The MCF-7ADR data provide evidence that this transition can occur in human breast cancer cells. Vimentin expression may provide useful insights into mechanisms of invasion and/or breast cancer cell progression.     

Hsa_circ_0092276 promotes doxorubicin resistance in breast cancer cells by regulating autophagy via miR-348/ATG7 axis

Previous study has confirmed that hsa_circ_0092276 is highly expressed in doxorubicin (DOX)-resistant breast cancer cells, indicating that hsa_circ_0092276 may be involved in regulating the chemotherapy resistance of breast cancer. Here we attempted to investigate the biological role of hsa_circ_0092276 in breast cancer. We first constructed DOX-resistant breast cancer cells (MCF-7/DOX and MDA-MB-468/DOX). The 50% inhibiting concentration of MCF-7/DOX and MDA-MB-468/DOX cells was significantly higher than that of their parental breast cancer cells, MCF-7 and MDA-MB-46. MCF-7/DOX and MDA-MB-468/DOX cells also exhibited an up-regulation of drug resistance-related protein MDR1. Compared with MCF-7 and MDA-MB-46 cells, hsa_circ_0092276 was highly expressed in MCF-7/DOX and MDA-MB-468/DOX cells. Hsa_circ_0092276 overexpression enhanced proliferation and the expression of LC3-II/LC3-I and Beclin-1, and repressed apoptosis of breast cancer cells. The effect of hsa_circ_0092276 up-regulation on breast cancer cells was abolished by 3-methyladenine (autophagy inhibitor). Hsa_circ_0092276 modulated autophagy-related gene 7 (ATG7) expression via sponging miR-384. Hsa_circ_0092276 up-regulation promoted autophagy and proliferation, and repressed apoptosis of breast cancer cells, which was abolished by miR-384 overexpression or ATG7 knockdown. In addition, LV-circ_0092276 transfected MCF-7 cell transplantation promoted autophagy and tumor growth of breast cancer in mice. In conclusion, our data demonstrate that hsa_circ_0092276 promotes autophagy and DOX resistance in breast cancer by regulating miR-348/ATG7 axis. Thus, this article highlights a novel competing endogenous RNA circuitry involved in DOX resistance in breast cancer.     

The influence of photodynamic therapy on apoptosis in human melanoma cell line

Melanoma is the most severe of all skin cancers as it may grow rapidly and metastasize. The application of photodynamic therapy (PDT) opens new perspectives in treatment of this cancer. Numerous studies suggest that the exposure of tumor cells to PDT can lead to cell death via two separate processes: apoptosis or necrosis. The aim of this study was to assess in vitro photodynamic therapy which induces apoptosis in the human Beidegr?m Melanoma (BM) cell line, using neutral comet assay. The cells were incubated with Photofrin II (15 microg/ml and 30 microg/ml) 4 h before and 3 h after irradiation for 5 or 10 min with the light intensity of 10 mW/cm2, using a lamp with red filter (632.8 nm). The percentage of apoptotic cells was significantly higher after PDT comparing to control cells. We observed 25% and 70% of apoptotic cells after shorter irradiation and treatment with 15 microg/ml and 30 microg/ml of Ph II, respectively. After longer irradiation, the respective values were 71.9% and 90%. The results suggest that induction of apoptosis is an important determinant of photodynamic sensitivity in the studied cell line and that some types of DNA damage are dependent on photosensitizer concentration and time of irradiation.     

Redox-responsive nanoparticles from the single disulfide bond-bridged block copolymer as drug carriers for overcoming multidrug resistance in cancer cells

Multidrug resistance (MDR) is a major impediment to the success of cancer chemotherapy. The intracellular accumulation of drug and the intracellular release of drug molecules from the carrier could be the most important barriers for nanoscale carriers in overcoming MDR. We demonstrated that the redox-responsive micellar nanodrug carrier assembled from the single disulfide bond-bridged block polymer of poly(ε-caprolactone) and poly(ethyl ethylene phosphate) (PCL-SS-PEEP) achieved more drug accumulation and retention in MDR cancer cells. Such drug carrier rapidly released the incorporated doxorubicin (DOX) in response to the intracellular reductive environment. It therefore significantly enhanced the cytotoxicity of DOX to MDR cancer cells. It was demonstrated that nanoparticular drug carrier with either poly(ethylene glycol) or poly(ethyl ethylene phosphate) (PEEP) shell increased the influx but decreased the efflux of DOX by the multidrug resistant MCF-7/ADR breast cancer cells, in comparison with the direct incubation of MCF-7/ADR cells with DOX, which led to high cellular retention of DOX. Nevertheless, nanoparticles bearing PEEP shell exhibited higher affinity to the cancer cells. The shell detachment of the PCL-SS-PEEP nanoparticles caused by the reduction of intracellular glutathione significantly accelerated the drug release in MCF-7/ADR cells, demonstrated by the flow cytometric analyses, which was beneficial to the entry of DOX into the nuclei of MCF-7/ADR cells. It therefore enhanced the efficiency in overcoming MDR of cancer cells, which renders the redox-responsive nanoparticles promising in cancer therapy.     

Cellular inactivation and antitumor efficacy of a new zinc phthalocyanine with potential use in photodynamic therapy

The aim of the present study was to evaluate the photodynamic efficacy of a novel phthalocyanine derivate 2,3,9,10,16,17,23,24-octakis[(N,N-dimethylamino) ethylsulfanyl]phthalocyaninatozinc(II) (referred here as S1) using MCF-7c3 human breast cancer cells and the LM2 adenocarcinoma subcutaneously implanted in Balb/c mice as experimental models. The S1-l-alpha-dimyristoyl-phosphatidylcholine liposome was selected as the best delivery system because it showed greater internalization into cells (35 nmol/10(6) cells), relative to other liposomes. After 3 h incubation S1 was partially localized in lysosomes, the compartment that represented its primary photodamage site. The S1 treated cultures also revealed a degree of mitochondrial morphology alteration. Indeed, S1 leads to photokilling of the cells with different efficacies indicating that cell photoinactivation was dependent on both the phthalocyanine concentration and the light dose applied. Analyses of morphology and nuclear condensation level indicated that some of the cells exposed to photodynamic therapy were undergoing apoptosis within 8h after treatment. To assess the in vivo effectiveness of S1, animals bearing tumors were treated with 0.2mg/kg S1 followed 24h later by 108 J cm(-2) light at 600-800 nm and 60 mW cm(-2),while other animals served as controls (no treatment, light alone, or S1 alone). All S1 treated tumors and none of the controls exhibited complete or partial responses, and these responses continued for the entire observation period of 12 days. Evaluation of tumor size showed that the treatment effectively delayed tumor growth. Light microscopy investigations of irradiated tumor specimens showed that S1 causes an early direct damage of malignant cells, largely via processes leading to random necrotic pathways.     

A combined treatment TNF-alpha/doxorubicin alleviates the resistance of MCF-7/Adr cells to cytotoxic treatment

The efficiency of anticancer therapy is often restricted by the development of drug resistance. Here, we report that the doxorubicin (DOX)-resistant MCF-7/Adr cells were more resistant to DOX-treatment than MCF-7 cells. However, an alternative treatment of DOX/TNF-alpha enhanced the cytotoxic effect in multidrug resistant MCF-7/Adr cell line. Treatment of cells with TNF-alpha following doxorubicin (DOX) resulted in a decrease of the activated Rel A/p65 in nuclei. Histone deacetylase 1 (HDAC1) was found to interact with Rel A/p65 in the complex, suggesting that HDAC1 is involved in mediating nuclear export of Rel A/p65. The combined treatment of TNF-alpha/DOX also resulted in a significant decrease of mRNA levels of anti-apoptotic genes, such as the cellular inhibitor of apoptosis-1 (c-IAP1), and the long isoform of B cell leukemia/lymphoma x gene (Bcl-xL), leading to efficient induction of caspase-8 cleavage and cell death. In previous work, we demonstrated that TNF-alpha promotes DOX-induced cell death and anti-cancer effect through downregulation of p21 in p53-deficient tumor cells. Thus, we proposed that alternative administration of TNF-alpha and DOX may be a new and efficient therapeutic strategy for patients that develop resistance to cytotoxic treatment.     

Interference with ER-α enhances the therapeutic efficacy of the selective CDK inhibitor roscovitine towards ER-positive breast cancer cells

In recent years many risk factors for the development of breast cancer that are linked to estrogens have been identified, and roscovitine (ROSC), a selective cyclin-dependent kinase (CDK) inhibitor, has been shown to be an efficient inhibitor of the proliferation of human breast cancer cells. Therefore, we have examined the possibility that interference with estrogen signaling pathways, using tamoxifen (TAM), a selective estrogen receptor modulator (SERM), could modulate the efficacy of treatment with ROSC. In conjunction with TAM, ROSC exhibited enhanced anti-proliferative activity and CDK inhibition, particularly in estrogen-dependent MCF-7 cells. The interaction between both drugs was synergistic. However, in ER-α-negative cells the interaction was antagonistic. Exposure of MCF-7 cells to ROSC abolished the activating phosphorylation of CDK2 and CDK7 at Ser(164/170). This in turn prevented the phosphorylation of the carboxyl-terminal repeat domain of RNA Polymerase II and ER-α at Ser(118), resulting in the down-regulation of the latter. Concomitantly, wt p53 was strongly activated by phosphorylation at Ser(46). Our results demonstrate that ROSC negatively affects the functional status of ER-α, making it potentially useful in the treatment of estrogen-dependent breast cancer cells.     

Encapsulation of methylene blue in polyacrylamide nanoparticle platforms protects its photodynamic effectiveness

The ability to prevent methylene blue (MB), a photosensitizer, from being reduced by plasma reductases will greatly improve its efficacy in photodynamic therapy (PDT) applications. We have developed a delivery approach for PDT by encapsulating MB using nanoparticle platforms (NPs). The 30-nm polyacrylamide-based NPs provide protection for the embedded MB against reduction by diaphorase enzymes. Furthermore, our data shows the matrix-protected MB efficiently induces photodynamic damage to tumor cells. The unprecedented results demonstrate the significant in vitro photodynamic effectiveness of MB when encapsulated within NPs, which promises to open new opportunities for MB in its in vivo and clinical studies.     

2DG enhances the susceptibility of breast cancer cells to doxorubicin

2DG causes cytotoxicity in cancer cells by disrupting thiol metabolism while Doxorubicin (DOX) induces cytotoxicity in tumor cells by generating reactive oxygen species (ROS). Here we examined the combined cytotoxic action of 2DG and DOX in rapidly dividing T47D breast cancer cells vs. slowly growing MCF-7 breast cancer cells. T47D cells exposed to the combination of 2DG/DOX significantly decreased cell survival compared to controls, while 2DG/DOX had no effect on MCF-7 cells. 2DG/DOX also disrupted the oxidant status of T47D treated cells, decreased intracellular total glutathione and increased glutathione disulfide (%GSSG) compared to MCF-7 cells. Lipid peroxidation increased in T47D cells treated with 2DG and/or DOX, but not in MCF-7 cells. T47D cells were significantly protected by NAC, indicating that the combined treatment exerts its action by increasing ROS production and disrupting antioxidant stores. When we inhibited glutathione synthesis with BSO, T47D cells became more sensitive to 2DG/DOX-induced cytotoxicity, but NAC significantly reversed this cytotoxic effect. Finally, 2DG/DOX, and BSO significantly increased the %GSSG in T47D cells, an effect which was also reversed by NAC. Our results suggest that exposure of rapidly dividing breast cancer cells to 2DG/DOX enhances cytotoxicity via oxidative stress and via disruptions to thiol metabolism.     

Photodynamic cell-kill analysis of breast tumor cells with a tamoxifen-pyropheophorbide conjugate

We hypothesized that estrogen receptor (ER) in hormone-sensitive breast cancer cells could be targeted for selective photodynamic killing of tumor cell with antiestrogen-porphyrin conjugates by combining the over-expression of ER in hormone-sensitive breast cancer cells and tumor-retention property of porphyrin photosensitizers. In this study we describe that a tamoxifen (TAM)-pyropheophorbide conjugate that specifically binds to ER alpha, caused selective cell-kill in MCF-7 breast cancer cells upon light exposure. Therefore, it is a potential candidate for ER-targeted photodynamic therapy of cancers (PDT) of tissues and organs that respond to estrogens/antiestrogens.     

Near-infrared dyes for six-color immunophenotyping by laser scanning cytometry

BACKGROUND: To adequately analyze the complexity of the immune system and reduce the required sample volume for immunophenotyping in general, more measurable colors for the discrimination of leukocyte subsets are necessary. Immunophenotyping by the laser scanning cytometer (LSC), a slide-based cytometric technology, combines cell detection based on multiple colors with their subsequent visualization without the need for physical cell sorting. In the present study, the filter setting of the LSC was adapted for the measurement of the far-red emitting dye cyanine 7 (Cy7), thereby increasing the number of measurable commercially available fluorochromes. METHODS: The optical filters of the LSC were replaced-photomultiplier (PMT) 3/allophycocyanin (APC): 740-nm dichroic long pass, and 670-/55-nm bandpass; PMT 4/Cy7: 810-/90-nm bandpass. Peripheral blood leukocytes were stained directly by fluorochrome-labeled antibodies or by indirect staining. The tandem dyes of Cy7 (phycoerythrin [PE]-Cy7, APC-Cy7) and the fluorochromes fluorescein isothiocyanate (FITC), PE, PE-Cy5, and APC were tested alone and in different combinations. RESULTS: With the new filter combination and tandem fluorochromes, Cy7 was measurable at 488-nm (argon laser) or 633-nm (helium-neon laser) excitation. Resolution was in the range of FITC for PE-Cy7 but approximately 30% lower for APC-Cy7; spillover into the respective donor fluorochrome channel for both tandem dyes was prominent. A six-color panel for leukocyte subtyping was designed. CONCLUSIONS: With this adaptation, it is possible to measure the tandem conjugates PE-Cy7 and APC-Cy7. This new setup opens the way for six-color immunophenotyping by LSC.     

Down-regulation of cellular FLICE-inhibitory protein (Long Form) contributes to apoptosis induced by Hsp90 inhibition in human lung cancer cells

Background

Breast cancer is the most common cancer in the Arab world and it ranked first among Saudi females. Doxorubicin (DOX), an anthracycline antibiotic is one of the most effective anticancer agents used to treat breast cancer. chronic cardiotoxicity is a major limiting factor of the use of doxorubicin. Therefore, our study was designed to assess the role of a natural product resveratrol (RSVL) on sensitization of human breast cancer cells (MCF-7) to the action of DOX in an attempt to minimize doxorubicin effective dose and thereby its side effects.

Methods

Human breast cancer cell line MCF-7, was used in this study. Cytotoxic activity of DOX was determined using (sulforhodamine) SRB method. Apoptotic cells were quantified after treatment by annexin V-FITC- propidium iodide (PI) double staining using flow-cytometer. Cell cycle disturbance and doxorubicin uptake were determined after RSVL or DOX treatment.

Results

Treatment of MCF-7 cells with 15 μg/ml RSVL either simultaneously or 24 h before DOX increased the cytotoxicity of DOX, with IC50 were 0.056 and 0.035 μg/ml, respectively compared to DOX alone IC50 (0.417 μg/ml). Moreover, flow cytometric analysis of the MCF-7 cells treated simultaneously with DOX (0.5 μg/ml) and RSVL showed enhanced arrest of the cells in G₀ (80%). On the other hand, when RSVL is given 24 h before DOX although there was more increased in the cytotoxic effect of DOX against the growth of the cells, however, there was decreased in percentage arrest of cells in G₀, less inhibition of DOX-induced apoptosis and reduced DOX cellular uptake into the cells.

Conclusion

RSVL treatment increased the cytotoxic activity of DOX against the growth of human breast cancer cells when given either simultaneously or 24 h before DOX.