Canady cold plasma conversion system treatment: An effective inhibitor of cell viability in breast cancer molecular subtypes

Progressive incidence and a pessimistic survival rate of breast cancer in women worldwide remains one of the most concerning topics. Progressing research indicates a potentially high effectiveness of use cold atmospheric plasma (CAP) systems. The undoubted advantage seems its simplicity in combination with other anti-cancer modalities. Following observed trend of studies, one inventory CAP system was applied to directly treat human breast cancer cell lines and culturing in two different Plasma Activated Media (PAM) for combined utilization. Proposed CAP treatments on MCF-10 A, MCF-7, and MDA-MB-231 cell lines were studied in terms of impact on cell viability by MTT assay. Disturbances in cell motility following direct and combined CAP application were assessed by scratch test. Finally, the induction of apoptosis and necrosis was verified with annexin V and propidium iodide staining. Reactive species generated during CAP treatment were determined based on optical emission spectrometry analysis along with colorimetric methods to qualitatively assess the NO2−, NO3−, H2O2, and total ROS with free radicals concentration. The most effective approach for CAP utilization was combined treatment, leading to significant disruption in cell viability, motility and mostly apoptosis induction in breast cancer cell lines. Determined CAP dose allows for mild outcome, showing insignificant harm for the non-cancerous MCF-10 A cell line, while the highly aggressive MDA-MB-231 cell line shows the highest sensitivity on proposed CAP treatment. Direct CAP treatment seems to drive the cells into the sensitive state in which the effectiveness of PAM is boosted. Observed anti-cancer response of CAP treatment was mostly triggered by RNS (mostly NO2− ions) and ROS along with free radicals (such as H2O2, OH•, O2-•, 1O2, HO2•). The combined application of one CAP source represent a promising alternative in the development of new and effective modalities for breast cancer treatment.

Background: Cold atmospheric plasma (CAP) is increasingly used in the field of oncology. Many of the mechanisms of action of CAP, such as inhibiting proliferation, DNA breakage, or the destruction of cell membrane integrity, have been investigated in many different types of tumors. In this regard, data are available from both in vivo and in vitro studies. Not only the direct treatment of a tumor but also the influence on its blood supply play a decisive role in the success of the therapy and the patient’s further prognosis. Whether the CAP influences this process is unknown, and the first indications in this regard are addressed in this study. Methods: Two different devices, kINPen and MiniJet, were used as CAP sources. Human endothelial cell line HDMEC were treated directly and indirectly with CAP, and growth kinetics were performed. To indicate apoptotic processes, caspase-3/7 assay and TUNEL assay were used. The influence of CAP on cellular metabolism was examined using the MTT and glucose assay. After CAP exposure, tube formation assay was performed to examine the capillary tube formation abilities of HDMEC and their migration was messured in separate assays. To investigate in a possible mutagenic effect of CAP treatment, a hypoxanthine-guanine-phosphoribosyl-transferase assay with non malignant cell (CCL-93) line was performed. Results: The direct CAP treatment of the HDMEC showed a robust growth-inhibiting effect, but the indirect one did not. The MMT assay showed an apparent reduction in cell metabolism in the first 24 h after CAP treatment, which appeared to normalize 48 h and 72 h after CAP application. These results were also confirmed by the glucose assay. The caspase 3/7 assay and TUNEL assay showed a significant increase in apoptotic processes in the HDMEC after CAP treatment. These results were independent of the CAP device. Both the migration and tube formation of HDMEC were significant inhibited after CAP-treatment. No malignant effects could be demonstrated by the CAP treatment on a non-malignant cell line.

We evaluated the association between dietary patterns and breast cancer (BC) subtypes among women from Northern Mexico. From a study of incident cases and population controls that was carried out from 2007 to 2011, a subsample of 509 cases matched 1:1 by age with 509 controls was selected. Information about expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 (HER2) was available from medical records to classify BC on luminal (ER + and/or PR+/HER2–), HER2+ (ER+/– and/or PR+/–/HER2+), or triple negative (ER– and PR–/HER2–). Dietary information was gathered using a semiquantitative food frequency questionnaire and a factor analysis was used to obtain dietary patterns. The association between each dietary pattern and BC molecular subtypes was assessed through conditional logistic regression models. Two dietary patterns were identified. The first (mainly characterized by meat, high fat, and sugary cereals) was positively associated with BC (odds ratio, OR = 12.62; 95% CI: 7.42, 21.45); the second (consisting of corn, legumes, and other vegetables) was inversely associated with BC (OR = 0.50; 95% CI: 0.40, 0.62). Both associations remained significant by BC molecular subtypes. These findings could contribute to the development of public health strategies for BC prevention.

Inconclusive epidemiological evidence suggests that diet quality indices may influence breast cancer (BC) risk; however, the evidence does not consider the molecular expression of this cancer. We aimed to evaluate if diet quality is related to molecular subtypes of BC, in women residing in Northern Mexico. This is a secondary analysis of 1,045 incident cases and 1,030 population controls from a previous case-control study, conducted between 2007 and 2011 in Northern Mexico. Information about the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 (HER2) was obtained from medical records to classify BC as luminal (ER + and/or PR+/HER2-), HER2+ (ER+/-and/or PR+/-/HER2+), or triple-negative (TN) (ER- and PR-/HER2-) cases. Food consumption was assessed with a semi-quantitative food frequency questionnaire. Diet quality was evaluated using the Mexican Diet Quality Index (MxDQI) and the Mexican Alternative Healthy Eating Index (MxAHEI). We used unconditional logistic regression models to estimate the association between Mexican diet quality indices and BC molecular subtypes. The MxDQI was related to lower odds of BC (ORT3vsT1=0.24; 95%CI: 0.18, 0.31). Similarly, MxAHEI was negatively associated with BC (ORT3vsT1=0.43; 95%CI: 0.34, 0.54). The associations of both indices remained significant in the ER + and ER- tumors, and in the BC luminal and HER2 + molecular subtypes, except in the TN molecular subtype for MxAHEI, which was not statistically significant. Our findings showed that MxDQI and MxAHEI were negatively associated with BC risk regardless of its molecular subtype.

Breast cancer is the leading cause of cancer mortality in females worldwide. Studies based on gene expression profiles have identified different breast cancer molecular subtypes, such as luminal A and B cells, cancer cells that are estrogen receptor (ER) and/or progesterone receptor (PR) positive, human epidermal growth factor 2 (HER2)-enriched cells, cancer cells that exhibit an overexpression of the oncogene HER2, and triple-negative cells, cancer cells that are negative for ER, PR and HER2 expression. Immunohistochemistry is the most common type of method used for the identification of these molecular subtypes, through the identification of specific cell receptors. The present study aimed to evaluate the ER, PR and HER2 receptor expression in human breast cancer cell lines, and to classify the corresponding molecular subtype comparing two alternative methods. In the present study, a panel of human mammary carcinoma cell lines: BT-20; Hs578T; MCF-7; MCF-7/AZ; MDA-MB-231; MDA-MB-468; SKBR3; and T47D were used. Immunohistochemical and immunocytochemistry assays were used to characterize the breast cancer subtypes of these cell lines according to the expression of ER, PR and HER2 receptors. The results revealed the molecular characterization of this panel of breast cancer cell lines, using the differential expression of classical and clinically used markers in concordance with previous studies. In addition, these data are important for additional in vitro studies of these specific receptors.

Background:Breast cancer is currently the leading cause of cancer morbidity and mortality among Algerian women. Molecular classification of breast cancer is an important factor for prognosis and clinical outcome.To our best knowledge,few studies have been reported on molecular subtypes of female breast cancer in Algeria.The objective of the present study was to evaluate age at diagnosis,distribution,prevalence, and association with menopausal status, histological type and histological grade of molecular subtypes of breast cancer in Algerian women.In addition, we screened for the prevalence of BRCA1 mutations in TNBC patients with and without family history of breast cancer. Methods: We analyzed breast cancers from cancer registries of academic medical oncology service of public hospital of Rouiba, anticancer center of Blida, and anticancer center of Batna.Breast cancers were diagnosed between 2008 and July 2013. ER (oestrogen receptor), PR (progesterone receptor) and HER2 (human epidermal growth factor receptor-2) status were obtained from medical records for 3014 patients.Breast cancer subtypes definitions were as follow:Luminal A (ER+ and/or PR+,HER2-), Luminal B (ER+ and/or PR+,HER2+),TNBC(ER-,PR-,HER2-), HER2+ (ER-,PR-,HER2+). 20 TNBC patients with family history of breast cancer and 33 sporadic TNBC women were screened by using PCR-direct sequencing for 3 common BRCA1 mutations c.83_84delTG, c181T>G and c.798_799delTT detected in previous studies in Algerian population. Results: The median age at diagnosis of 3014 patients with breast cancer was 48.5 years. The prevalence of the Luminal A, TNBC, Luminal B and HER2+ were 50.59%,20.80%, 19.67% and 8.92%,respectively.Luminal A, Luminal B, TNBC and HER2+ subtypes were more prevalent among premenopausal women (29.24%, 13.26%, 12.19% and 4.80%) compared with postmenopausal women (21.74%, 7.31%, 7.89% and 3.87%). Invasive Ductal Carcinoma was the most prevalent histological type in all breast cancer subtypes at any age. Tumors with histological grade 2 were more prevalent in younger patients for the four breast cancer subtypes. High prevalence of histological grade 3 was showed in older patients. The BRCA1 mutation c.83_84delTG was detected in one young TNBC patient with a family history of cancer. Conclusion: For the first time, we report the distribution of molecular subtypes of breast cancer and some clinical characteristics in a large cohort of Algerian women. In our current study, the median age of diagnosis for all breast cancer subtypes was younger than the average age in Europe and America.Interestingly, TNBC subtype was the second most prevalent in our patients,and the prevalence of luminal subtype was lesser than reported in white women with breast cancer in Europe and America. The high prevalence of TNBC reported here could be linked to Sub-Saharan African genetic elements of Algerian population.Screening for BRCA1 mutations in large series of TNBC patients is ongoing. Citation Format: Farid Cherbal, Hadjer Gaceb, Chiraz Mehemmai, Rabah Bakour, Kada Boualga, Wassila Benbrahim, Hassen Mahfouf. Molecular subtypes of breast cancer in Algeria: a population-based study of 3014 women. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3833. doi:10.1158/1538-7445.AM2014-3833

Cold atmospheric plasma reduces demodex count on the face comparably to topical ivermectin, as measured by reflectance confocal microscopy.

Cold Atmospheric Plasma Suppresses Ovarian Cancer Cell Activity With Concurrent Secretion Of Heat Shock Protein 27 Affecting Monocyte Fate

Epigenetic Regulation of Cancer Stem Cell Genes in Triple-Negative Breast Cancer

Breast cancer exists in multiple subtypes some of which still lack a targeted and effective therapy. Cold atmospheric plasma (CAP) has been proposed as an emerging anti-cancer treatment modality. In this study, we investigated the effects of direct and indirect CAP treatment driven by the advantageous nanosecond pulsed discharge on breast cancer cells of different malignant phenotypes and estrogen receptor (ER) status, a major factor in the prognosis and therapeutic management of breast cancer. The main CAP reactive species in liquid (i.e. H2O2, ) and gas phase were determined as a function of plasma operational parameters (i.e. treatment time, pulse voltage and frequency), while pre-treatment with the ROS scavenger NAC revealed the impact of ROS in the treatment. CAP treatment induced intense phenotypic changes and apoptosis in both ER+ and ER- cells, which is associated with the mitochondrial pathway as evidenced by the increased Bax/Bcl-2 ratio and cleavage of PARP-1. Interestingly, CAP significantly reduced CD44 protein expression (a major cancer stem cell marker and matrix receptor), while differentially affected the expression of proteases and inflammatory mediators. Collectively, the findings of the present study suggest that CAP suppresses breast cancer cell growth and regulates several effectors of the tumor microenvironment and thus it could represent an efficient therapeutic approach for distinct breast cancer subtypes.

Cold Atmospheric Plasma Selectively Induces Apoptosis In Renal Adenocarcinoma

The oxidative species generated by cold atmospheric plasma (CAP) treatment can impact the plant stress response system. We hypothesized that this response is not limited to the site of CAP application and it is transmitted through the plant. The resulting stress response can influence the plant microbiome on the intact plant. These hypotheses were tested by the application of CAP to live sweet basil (Ocimum basilicum var. Kiera). A single upper leaf of the plant underwent a 60 s CAP treatment at three different wattage intensity levels. Reactive oxygen species (ROS) generation in directly treated leaves and leaves in the vicinity of the treatment site (i.e., one, two, or three nodes away) was measured using the fluorescein degradation assay (ex/em: 485/525). Leaves directly exposed to CAP showed a marked increase in ROS production. Interestingly, basil leaves not directly treated by CAP also showed a significant (p < 0.05) increase in ROS generation compared to untreated control, extending to the two nearest nodes from the treatment site in all plants tested. The leaf microbiomes were evaluated using 16S rRNA gene sequencing. CAP appeared to drive restructuring of the leaf microbiota profiles, despite maintaining a similar α‐diversity. CAP treatment intensity led to significant differences (p < 0.05) in the relative abundances of a variety of dominant bacterial families (e.g., Psuedomonadaceae and Streptomycetaceae) and phyla, and the effects on certain taxa were dependent on leaf distance from the treatment site. CAP's ability to restructure plant microbiota may have applications to improve produce microbial safety and shelf‐life.Practical ApplicationCold atmospheric plasma induces a stress response in a living plant beyond the site of application. This response includes an increase in the production of reactive oxygen species that can trigger pathways to enhance the production of phytochemicals. CAP treatment also alters the microbial community profile, possibly through plant stress response. Results from this study can be useful in developing CAP treatment of intact plant for improved growth, production of health‐benefiting phytochemicals, and managing its microbiota.

Cold Atmospheric Plasma (CAP) is a promising novel method for biofilm inactivation as log-reduction values up to 4.0 log10 (CFU/cm2) have been reported. Nevertheless, as the efficacy of CAP itself is not sufficient for complete inactivation of mature biofilms, the hurdle technology could be applied in order to obtain higher combined efficacies. In this study, CAP treatment was combined with a mild hydrogen peroxide (H2O2) treatment for disinfection of 1 and 7 day(s) old Listeria monocytogenes and Salmonella Typhimurium biofilms. Three different treatment sequences were investigated in order to determine the most effective treatment sequence, i.e., (i) first CAP, then H2O2, (ii) first H2O2, then CAP, and (iii) a simultaneous treatment of CAP and H2O2. Removal of the biofilm, induction of sub-lethal injury, and H2O2 breakdown due to the presence of catalase within the biofilms were investigated in order to comment on their possible contribution to the combined inactivation efficacy. Results indicated that the preferred treatment sequence was dependent on the biofilm forming species, biofilm age, and applied H2O2 concentration [0.05 or 0.20% (v/v)]. At the lowest H2O2 concentration, the highest log-reductions were generally observed if the CAP treatment was preceded by the H2O2 treatment, while at the highest H2O2 concentration, the opposite sequence (first CAP, then H2O2) proved to be more effective. Induction of sub-lethal injury contributed to the combined bactericidal effect, while the presence of catalase within the biofilms resulted in an increased resistance. In addition, high log-reductions were partially the result of biofilm removal. The highest overall log-reductions [i.e., up to 5.42 ± 0.33 log10 (CFU/cm2)] were obtained at the highest H2O2 concentration if CAP treatment was followed by H2O2 treatment. As this resulted in almost complete inactivation of the L. monocytogenes and S. Typhimurium biofilms, the combined treatment of CAP and H2O2 proved to be a promising method for disinfection of abiotic surfaces.

Exploring the Potential of Breast Microbiota as Biomarker for Breast Cancer and Therapeutic Response

The purpose of this study is to evaluate sublethal cold atmospheric plasma (CAP) treatment of filamentous fungal pathogen susceptibility to commonly used antifungal drugs in vitro. Response to CAP in combination with voriconazole, fluconazole, amphotericin B, and caspofungin was evaluated in Aspergillus flavus and Fusarium keratoplasticum conidia and mycelium; conidial response to fluconazole was also assessed in three strains of F. falciforme. Conidial susceptibility to antifungal drugs alone or in combination with CAP was assessed using a modified CLSI broth microdilution assay with MIC determination and colony-forming unit (CFU) enumeration. Mycelial viability and biofilm thickness changes in response to antifungal drugs alone or in combination with CAP were assessed over 24 hours post treatment. CAP enhanced antifungal drug efficacy against all fungal species, though effects differed by drug and growth form. CAP enhanced antifungal drug susceptibility in conidia, with the strongest effect observed for F. keratoplasticum conidia, where caspofungin MIC decreased fourfold (from 16 to 4-8 μg/mL) and sensitivity to fluconazole, which exerted no effect in absence of CAP, was restored when combined with sublethal CAP treatment. In A. flavus, CAP lowered the MIC of voriconazole (from 0.25 to 0.06-0.125 μg/mL) but increased the MIC of amphotericin B (from 4 to >4 μg/mL), despite reductions in viable cell counts. Differential responses to CAP and fluconazole were observed across three strains of F. falciforme, suggesting variability in CAP response. In treated biofilms, CAP alone initially reduced mycelial viability and biofilm thickness, but partial recovery of the fungus was observed over time in most cases. When combined with antifungal drugs, CAP significantly enhanced reduction of mycelial viability and thickness beyond antifungal treatment alone. In F. keratoplasticum biofilms, the combination of CAP and antifungal drugs produced sustained reductions in mycelial viability and biofilm thickness, whereas A. flavus biofilms were more resistant to CAP treatment and exhibited consistent recovery after 24 hours.