Rhodamine Staining Research Articles

Ciprofol Ameliorates Myocardial Ischemia/Reperfusion Injury by Inhibiting Ferroptosis Through Upregulating HIF-1α.

Ciprofol is a novel intravenous anesthetic that has been increasingly used in clinical anesthesia and sedation. Studies suggested that ciprofol reduced oxidative stress and inflammatory responses to alleviate cerebral ischemia/reperfusion (I/R) injury, but whether ciprofol protects the heart against I/R injury and the mechanisms are unknown. Herein, we assessed the effects of ciprofol on ferroptosis during myocardial I/R injury. Experimental models of myocardial I/R injury in mice (ischemia for 30min and reperfusion for 24h) and hypoxia/reoxygenation (H/R) injury in H9c2 cardiomyocytes (hypoxia for 6h followed by 6h of reoxygenation) were established. Ciprofol was used prior to ischemia or hypoxia. Echocardiography, myocardial TTC staining, HE staining, DAB-enhanced Perl's staining, transmission electron microscopy, FerroOrange staining, Liperfluo staining, JC-1 staining, Rhodamine-123 staining, DCFH-DA staining, and Western blot were performed. Cell viability, serum cardiac enzymes, and oxidative- and ferroptosis-related biomarkers were measured. HIF-1α siRNA transfection and the specific inhibitor BAY87-2243 were utilized for mechanistic investigation. Ciprofol treatment reduced myocardial infarct area and myocardium damage, alleviated oxidative stress and mitochondrial injury, suppressed Fe2+ accumulation and ferroptosis, and improved cardiac function in mice with myocardial I/R injury. Ciprofol also increased cell viability, attenuated mitochondrial damage, and reduced intracellular Fe2+ and lipid peroxidation in cardiomyocytes with H/R injury. Ciprofol enhanced the protein expression of HIF-1α and GPX4 and reduced the expression of ACSL4. Specifically, the protective effects of ciprofol against I/R or H/R injury were abolished by downregulating the expression of HIF-1α using siRNA transfection or the inhibitor BAY87-2243. Ciprofol ameliorated myocardial I/R injury in mice and H/R injury in cardiomyocytes by inhibiting ferroptosis via the upregulation of HIF-1α expression.

Biosynthesis and Characterization of Silver Nanoparticles Using Cleistanthus collinus; Investigation on Molecular Mechanism of Apoptosis Cell Death Against Lung Cancer Cells

Background: The discovery of novel molecular and cellular signaling pathways for cancer medicines would enhance the efficacy of cancer therapy. In the current study, we made an effort to explore the mitochondrial-mediated apoptosis cell death signaling pathway in A549 lung cancer cells by silver nanoparticles (AgNPs) synthesized from leaf extract of Cleistanthus collinus. Methods: In-depth, A549 lung cancer cells were treated with AgNPs, and further studies such as HOECHST 33342, AO/EB, Rhodamine-123 staining, flowcytometry, RT-PCR, and Western blotting techniques (24, 48, and 72 h) evidenced apoptosis pathway in cancer cells. Results: Indeed, the microscopic studies proved that AgNPs-treated lung cancer cells appeared with cell shrinkage, membrane swelling, and apoptotic body formation whereas the untreated cells (control) failed to show any morphological consequences. The flow cytometry analysis revealed the G2/M phase of cell cycle arrest by cells were accumulated in large numbers. The RT-PCR data confirmed the expression (Bax and p53) and suppression (Bcl 2) apoptosis-responsive genes in cancer cells. Finally, immunoblotting proved the increased expression of cytochrome c, initiator caspase 9, and executioner caspase 3, which are effective proteins of intrinsic apoptosis activation. Conclusion: Ultimately, the overall present investigations confirmed the mitochondrial signaling pathway played a key role in inducing apoptosis in A549 cancer cell death and demonstrated that AgNPs are novel therapeutic agents for cancer nanomedicine

Ameliorative impacts of carvacrol on DNA integrity, oxidative stress, and sperm quality in asthenozoospermic infertile individuals during cryopreservation.

Nowadays, the cryopreservation process can produce reactive oxygen species (ROS), which cause damage to the motility, cell membrane, and DNA integrity of sperm. This study is aimed at evaluating the impact of carvacrol, as a powerful antioxidant, on sperm features and improving oxidative stress throughout the cryopreservation procedure. In this prospective study, semen samples from 25 patients with asthenozoospermia were separated into three groups: fresh, freezing, and freezing + carvacrol (a dose of 100µM). The subsequent parameters were evaluated using standard methods in all three groups: sperm motility according to WHO criteria, sperm morphology using Papanicolaou staining, sperm viability with eosin-nigrosin staining, DNA integrity with acridine orange staining, levels of antioxidant enzymes (catalase, glutathione, and superoxide dismutase), total antioxidant capacity (TAC), and malondialdehyde (MDA) using ELISA. Also, DNA fragmentation was analyzed by the SDFA kit, and mitochondrial membrane potential (MMP) was assessed by rhodamine staining. Average sperm viability, motility, mitochondrial membrane potentiality, integrity of sperm membrane, and antioxidant enzyme levels are meaningfully reduced in the freezing group in contrast to the control group. The freezing group showed a meaningful rise in the mean MDA levels and DNA fragmentation compared to the control group. In the freezing group supplemented with carvacrol, a meaningful rise could be visible in mean percentages of viability, motility, and antioxidant enzyme levels, whereas mean levels of MDA and DNA fragmentation meaningfully declined in contrast to the freezing group. The results demonstrate that carvacrol, a potent antioxidant, offers significant protection against the loss generated by the freeze-thaw procedure, thereby improving sperm quality.

Antiproliferative and Apoptotic Potential of Chicoric Acid, a Major Phytoconstituent of Cichorium intybus, against Colon Cancer Cells: A Possible Inhibition of CDC25 Phosphatases

Background: Over the years, plants have been utilized worldwide for the treatment of cancer in the traditional system of medicine, notably in the majority of developing nations. The use of chemotherapeutic drugs is frequently associated with lethal side effects when treating cancer. Therefore, the panacea to the vicious side effects linked to synthetic pharmaceuticals is the alternative usage of widely accessible and affordable therapeutic approaches. Chicoric acid, a major constituent of Cichorium intybus, has recently been shown to exhibit antioxidant, anti-inflammatory, antidiabetic, antiviral, neuroprotective, hepatoprotective, and anticancer properties. Purpose: Except for a few preliminary studies, no detailed study on the chemotherapeutic efficacy of chicoric acid against cancer cells has been done till now. Therefore, the aim of this study is to assess the potential of chicoric acid against colon cancer cells. Methods: Different in vitro assays like MTT, DAPI staining, rhodamine staining, H2DCFDA staining, caspase activity, and cell analysis were performed on colon cancer HCT116 cells to assess their growth inhibitory properties. Results: According to the results, chicoric acid dose-dependently suppressed the growth of colon cancer HCT116 cells by inducing cell cycle arrest at G0/G1 phase and apoptosis via both intrinsic and extrinsic pathways. In silico findings unveiled the cell cycle regulatory capacity of chicoric acid through binding CDC25 phosphatases, implying its potential as an antiproliferative agent capable of suppressing cell growth. Conclusion: Collectively, the results suggested strong chemopreventive potential of chicoric acid against colon cancer cells. Thus, chicoric acid may turn out to be an excellent anticancer agent, which may be explored in the future for the therapeutic drug development against cancer.

Antiproliferative Effects of Methanolic Fruit Extract of Solanum xenthocarpum (L.) on Human Breast Cancer Cells.

Solanum xanthocarpum, a perennial herb native to India, contains steroidal glycoalkaloids with notable anticancer properties. This study investigated the antioxidant and antiproliferative effects ofmethanolic fruit extract of S. xanthocarpum on human breast cancer cells (MDA-MB-231). Phytochemical screening and LC-HRMS analysis confirmedpresence of various primary and secondary metabolites. Antioxidant activity was assessed through DPPH, ABTS radical scavenging, reducing power, and phosphomolybdate assays. The extract demonstrated significant antioxidant potential with EC50 values of 60.10 ± 0.88 µg/mL (DPPH) and 392.29 ± 3.93 µg/mL (ABTS). Cytotoxicity against MDA-MB-231 cells was evaluated via morphological analysis, MTT assays, and IC50 determination (24.19 ± 0.56 µg/L). Apoptosis was confirmed using dual staining techniques (AO/EB, Hoechst 33342/PI, DAPI), revealing condensed nuclei, apoptotic bodies, and reduced mitochondrial membrane potential, as indicated by Rhodamine staining. Additionally, increased reactive oxygen species (ROS) levels were observed using H2-DCF-DA staining. The total phenolic and flavonoid contents of the extract were 127.78 ± 3.547 mg GAE/g and 98.06 ± 4.289 mg QE/g, respectively. These findings suggest that the methanolic fruit extract of S. xanthocarpum possesses strong antioxidant and anticancer activities, indicating its potential role in cancer treatment. Further studies are warranted to explore its bioactive compounds for developing novel anticancer therapies.

An optimization method for measuring the stomata in cassava (Manihot esculenta Crantz) under multiple abiotic stresses.

As a gateway for gas exchange, pores regulate the transport of air and water in carbon assimilation, respiration, and transpiration to quickly adapt to environmental changes. Therefore, the study of stomatal movement characteristics of plants is helpful to strengthen the understanding of the mechanism of plant response to multi-environmental stress, and can improve the function of plant resistance to stresses. The stomatal movement of Arabidopsis leaves was observed by staining the stomata with rhodamine 6G, but this method has not been reported in other plant leaf stomata studies. Taking cassava as an example, the correlation between cassava stomatal movement and cassava response to stress was observed by using and improving the staining method. Rhodamine 6G is a biological stain widely used in cell biology and molecular biology. It was found that 1 μM rhodamine 6G could stain the stomata of cassava without affecting stomatal movement (n = 109, p < 0.05). In addition, we proposed that stomata fixed with 4% concentration of formaldehyde after staining were closest to the stomatal morphology of cassava epidermis, so as to observe stomatal movement under different environmental stresses more accurately. Previous methods of measuring stomatal pore size by autofluorescence of cell wall needs to fix the cells for 6 h, but Rhodamine staining can only be observed in 2 min, which greatly improves the experimental efficiency. Compared with the traditional exfoliation method (e.g., Arabidopsis), this method can reduce the damage of the leaves and observe the stomata of the whole leaves more completely, so that the experimental results are more complete. In addition, the method enables continuous leaf processing and observation. Using this method, we further compared four different cassava varieties (i.e., KU50, SC16, SC8, and SC205) and found that there are differences in stomatal density (SD) among cassava varieties, and the difference in the SD directly affects the stress resistance of cassava (n = 107, p < 0.001). This finding has important implications for studying the mechanism of plant response to environmental stress through stomata.

Native and modified starch nano-crystals loaded with doxorubicin mediated apoptosis in human breast cancer MCF7 cells

The present study was aimed at evaluating the effect of doxorubicin (Dox) loaded nanocrystals starch in human breast cancer MCF7 cells. A model for breast cancer, MCF7 cells, were treated with Dox loaded in S (native starch), NS (starch nanocrystal), ANS (acetylated starch nanocrystal) extracted from Oryza glaberrima Steud and compared with Dox (diluted in water). MTT assay, Hoechst33342, H2DCFDA, Live Death, and Rhodamine123 staining, and Western Blot were used to detect different signaling protein expressions and apoptosis. Hoechst33342 staining showed cellular apoptosis of MCF7 after treating with DOX loaded S, NS, ANS or Dox alone. Intracellular ROS levels, p53 and Bax/ Bcl2 expressions, and decrease in mitochondrial membrane potential were also higher in MCF7 cells treated with Dox loaded S, NS, ANS or Dox alone. To ensure Dox loaded S, NS or ANS did not cause any cellular damages to normal cells, HEK-293 kidney cells were used as control. Dox loaded S, NS, or ANS were as effective formulations as Dox alone to decrease progression and metastasis of breast cancer in MCF7 cells. This study provides a novel approach for further investigation since Dox loaded starch nanocrystals are effective and can prevent side effects of cardiotoxicity when using Dox alone.

Chitosan-Polyethylene Glycol Inspired Polyelectrolyte Complex Hydrogel Templates Favoring NEO-Tissue Formation for Cardiac Tissue Engineering.

Neo-tissue formation and host tissue regeneration determine the success of cardiac tissue engineering where functional hydrogel scaffolds act as cardiac (extracellular matrix) ECM mimic. Translationally, the hydrogel templates promoting neo-cardiac tissue formation are currently limited; however, they are highly demanding in cardiac tissue engineering. The current study focused on the development of a panel of four chitosan-based polyelectrolyte hydrogels as cardiac scaffolds facilitating neo-cardiac tissue formation to promote cardiac regeneration. Chitosan-PEG (CP), gelatin-chitosan-PEG (GCP), hyaluronic acid-chitosan-PEG (HACP), and combined CP (CoCP) polyelectrolyte hydrogels were engineered by solvent casting and assessed for physiochemical, thermal, electrical, biodegradable, mechanical, and biological properties. The CP, GCP, HACP, and CoCP hydrogels exhibited excellent porosity (4.24 ± 0.18, 13.089 ± 1.13, 12.53 ± 1.30 and 15.88 ± 1.10 for CP, GCP, HACP and CoCP, respectively), water profile, mechanical strength, and amphiphilicity suitable for cardiac tissue engineering. The hydrogels were hemocompatible as evident from the negligible hemolysis and RBC aggregation and increased adsorption of plasma albumin. The hydrogels were cytocompatible as evident from the increased viability by MTT (>94% for all the four hydrogels) assay and direct contact assay. Also, the hydrogels supported the adhesion, growth, spreading, and proliferation of H9c2 cells as unveiled by rhodamine staining. The hydrogels promoted neo-tissue formation that was proven using rat and swine myocardial tissue explant culture. Compared to GCP and CoCP, CP and HACP were superior owing to the cell viability, hemocompatibility, and conductance, resulting in the highest degree of cytoskeletal organization and neo-tissue formation. The physiochemical and biological performance of these hydrogels supported neo-cardiac tissue formation. Overall, the CP, GCP, HACP, and CoCP hydrogel systems promise novel translational opportunities in regenerative cardiology.

Fabrication of a 3D bioprinting model for posterior capsule opacification using GelMA and PLMA hydrogel-coated resin.

Posterior capsule opacification (PCO) remains the predominant complication following cataract surgery, significantly impairing visual function restoration. In this study, we developed a PCO model that closely mimics the anatomical structure of the crystalline lens capsule post-surgery. The model incorporated a threaded structure for accurate positioning and observation, allowing for opening and closing. Utilizing 3D printing technology, a stable external support system was created using resin material consisting of a rigid, hollow base and cover. To replicate the lens capsule structure, a thin hydrogel coating was applied to the resin scaffold. The biocompatibility and impact on cellular functionality of various hydrogel compositions were assessed through an array of staining techniques, including calcein-AM/PI staining, rhodamine staining, BODIPY-C11 staining and EdU staining in conjunction with transwell assays. Additionally, the PCO model was utilized to investigate the effects of eight drugs with anti-inflammatory and anti-proliferative properties, including 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), THZ1, sorbinil, 4-octyl itaconate (4-OI), xanthohumol, zebularine, rapamycin and caffeic acid phenethyl ester, on human lens epithelial cells (HLECs). Confocal microscopy facilitated comprehensive imaging of the PCO model. The results demonstrated that the GelMA 60 5% + PLMA 2% composite hydrogel exhibited superior biocompatibility and minimal lipid peroxidation levels among the tested hydrogels. Moreover, compared to using hydrogel as the material for 3D printing the entire model, applying surface hydrogel spin coating with parameters of 2000 rpm × 2 on the resin-based 3D printed base yielded a more uniform cell distribution and reduced apoptosis. Furthermore, rapamycin, 4-OI and AICAR demonstrated potent antiproliferative effects in the drug intervention study. Confocal microscopy imaging revealed a uniform distribution of HLECs along the anatomical structure of the crystalline lens capsule within the PCO model, showcasing robust cell viability and regular morphology. In conclusion, the PCO model provides a valuable experimental platform for studying PCO pathogenesis and exploring potential therapeutic interventions.

Spatial variations in the osteocyte lacuno-canalicular network density and analysis of the connectomic parameters.

Osteocyte lacuno-canalicular network (LCN) is comprised of micrometre-sized pores and submicrometric wide channels in bone. Accumulating evidence suggests multiple functions of this network in material transportation, mechanobiological signalling, mineral homeostasis and bone remodelling. Combining rhodamine staining and confocal laser scanning microscopy, the longitudinal cross-sections of six mouse tibiae were imaged, and the connectome of the network was quantified with a focus on the spatial heterogeneities of network density, connectivity and length of canaliculi. In-vivo loading and double calcein labelling on these tibiae allowed differentiating the newly formed bone from the pre-existing regions. The canalicular density of the murine cortical bone varied between 0.174 and 0.243 μm/μm3, and therefore is three times larger than the corresponding value for human femoral midshaft osteons. The spatial heterogeneity of the network was found distinctly more pronounced across the cortex than along the cortex. We found that in regions with a dense network, the LCN conserves its largely tree-like character, but increases the density by including shorter canaliculi. The current study on healthy mice should serve as a motivating starting point to study the connectome of genetically modified mice, including models of bone diseases and of reduced mechanoresponse.

Inhibitory Effect of Heparin/Polyarginine/Hyaluronic Acid Complex on Coronary Artery Restenosis by Loading Vascular Endothelial Growth Factor

This research was aimed to investigate the inhibitory effect of heparin/polyarginine/hyaluronic acid (HPHA) complex loaded with vascular endothelial growth factor (VEGF) on coronary artery restenosis (CAR). Dopamine was grafted to obtain dopamine-hyaluronic acid (DPA) composite material, and heparin/polyarginine (HP) nanoparticles (NPs) loaded with VEGF were obtained through electrostatic binding (ESB). The HP NPs loaded with VEGF mixed with hyaluronic acid (HA) were mixed in a 10:1 volume ratio to obtain a composite solution, namely HPHA complex. Next, the sodium metaperiodate was added to form a hydrogel. The samples were characterized using a zeta potential (ZP) analyzer, transmission electron microscopy (TEM), Fourier transform infrared spectrometer (FTIR), and water contact angle (WCA). Enzyme-linked immunosorbent assay (ELISA) and toluidine blue O (TBO) staining were employed to assess the immobilization and release of VEGF and heparin. Rhodamine staining was adopted to evaluate the adhesion of platelets, fibrinogen, macrophages, and umbilical artery smooth muscle cells (UASMCs). Furthermore, CCK-8 assay was performed to evaluate the adhesion and proliferation of endothelial cells (ECs). The results revealed that compared to 0 mg/L, the particle size (PS), polydispersity index (PDI), and θ angle decreased, and the absolute value of ZP increased at 100, 200, 300, and 400 mg/L. The cumulative release (CR) of VEGF at 200 mg/L was higher to that at 0 mg/L, while that of heparin was lower (P &lt;0.05). The platelet surface coverage rate (PSCR), relative platelet activation rate (RPAR), and fibrinogen coverage rate (FCR) at 10 and 20 days were lower and exhibited great difference to those at 0 mg/L (P &lt;0.05). Compared to groups II and III, group I presented a markedly higher number of macrophages and UASMCs on the first and third days of culture (P &lt;0.05). 3 days after culture, adhesion and proliferation of ECs in groups U, I, and K increased greatly (P &lt;0.05). In conclusion, the HPHA complex prepared from HP NPs loaded with VEGF and HA in this research exhibited good stability, uniformity, hydrophilicity, and blood compatibility. It effectively blocked the adhesion of macrophages and UASMCs and promoted the proliferation of ECs, thus conforming to the mechanism of inhibiting CAR after coronary stent intervention.

Anticancer activity of zinc-nickel nanocomposite in lung cancer PC14 cells via modulation of apoptosis and P13K/mTOR pathway

Advanced innovations for combating variants of aggressive cancer and overcoming drug resistance are desired. In cancer treatment, nanoparticles (NPs) have the capacity to specifically and compellingly activate apoptosis of cancer cells. There is also a pressing need to develop innovative anti-cancer therapeutics, and recent research suggests that ZnO nanoparticles hold great potential.The current exploration was designed to fabricate the zinc-nickel nanomaterial (Zn-Ni NPs) and examine its in vitro anticancer effects on lung cancer cell. In ultraviolet–visible spectroscopy (UV–Vis), the bands at the wavelengths of 219, 265, 397, and 458 nm are related to the surface plasmon resonance of the synthetic Zn-Ni NPs. In fourier transform-infrared spectroscopy (FT-IR), the peaks of 449, 618, 659, 662, and 693 can be assigned to Zn-O or Ni-O bonds. The cytotoxicity of the Zn-Ni NPs (1 to 1000 µg/mL) were studied by the MTT assay and the IC50 dose for Zn-Ni NPs were found at 77 µg/mL. The different fluorescent staining assays such as Dichloro-dihydro-fluorescein diacetate, Rhodamine-123, and dual staining were performed to investigate the influence of Zn-Ni NPs on the apoptosis, matrix metalloproteinase status, and reactive oxygen species accumulation in the PC14 cells. The status of inflammatory markers such as IL-6, COX-2, TNF-α, and NF-κB in the PC14 cells were studied. The increased dosage of Zn-Ni NPs were effective repressed the growth of PC14 cells. The PC14 cells treated with Zn-Ni NPs revealed a significantly fewer proliferating cells and more apoptotic cells when compared with control. The Zn-Ni NPs also inhibited the phosphorylation of mTOR and Akt, reducing tumor volume and weight. PC14 cells phosphorylated mTOR, cyclin D1, Akt, and PI3K in response to Zn-Ni NPs. The findings were revealed that the fabricated Zn-Ni NPs considerably increased the ROS accumulation and apoptosis in the PC14 cells. Zn-Ni NPs also enhanced mitochondrial apoptosis through Bcl-2 protein-dependent signaling. In conclusion, Zn-Ni NPs might be promising candidate for lung cancer treatment.

Some 2-[4-(1H-benzimidazol-1-yl) phenyl]-1H-benzimidazole derivatives overcome imatinib resistance by induction of apoptosis and reduction of P-glycoprotein activity.

Imatinib (IMA) is a tyrosine kinase inhibitor (TKI) introduced for the chronic myeloid leukemia (CML) therapy. Emergence of IMA resistance leads to the relapse and failure in CML therapy. Benzimidazole is a heterocyclic organic compound which is widely investigated for the development of anticancer drugs. In this study, we aimed to explore the anticancer effects of some 2-[4-(1H-benzimidazol-1-yl) phenyl]-1H-benzimidazole derivatives on K562S (IMA-sensitive) and K562R (IMA-resistant) cells. To analyze the cytotoxic and apoptotic effects of the compounds, K562S, K562R, and L929 cells were exposed to increasing concentrations of the derivatives. Cytotoxic effects of compounds on cell viability were analyzed with MTT assay. Apoptosis induction, caspase3/7 activity were investigated with flow cytometry and BAX, BIM, and BAD genes expression levels were analyzed with qRT-PCR. Rhodamine123 (Rho-123) staining assays were carried out to evaluate the effect of compounds on P-glycoprotein (P-gp) activity. The hit compounds were screened using molecular docking, and the binding preference of each compounds to BCR-ABL protein was evaluated. Our results indicated that compounds triggered cytotoxicity, caspase3/7 activation in K562S and K562R cells. Rho-123 staining showed that compounds inhibited P-gp activity in K562R cells. Overall, our results reveal some benzimidazole derivatives as potential anticancer agents to overcome IMA resistance in CML.

Abstract 5311: Auranofin sensitizes paclitaxel induced apoptotic cell death through oxidative mechanism in breast cancer cells

Abstract Breast cancer cells exhibit several basal redox systems, thus poorly responding to oxidative cell death. These intrinsic redox statuses capitalize on the efficacy of chemotherapeutic drugs. Auranofin, an FDA-approved gold compound documented with effective pharmacokinetics and safety profiles in humans, has recently been repurposed for anticancer activity. This study examined the paclitaxel sensitizing effect of auranofin by targeting redox balance in the MDA-MB-231 and MCF-7 breast cancer cell lines. We observed that auranofin inhibits thioredoxin reductase activity (TrxR) in these breast cancer cells and collapses the cellular antioxidants status, including the reduced activities of superoxide dismutase, catalase, and glutathione peroxidase. Furthermore, it has been noticed that auranofin augmented paclitaxel mediated cytotoxicity in a concentration dependant manner in both the MDA-MB-231 and MCF-7 cell lines. Furthermore, auranofin increased intracellular ROS levels (DCFH-DA staining) and altered mitochondrial membrane potential (Rhodamine-123 staining) in these breast cancer cell lines. The proapoptotic markers such as p53, Bax, and caspase-3 and caspase-9 expressions were higher in auranofin plus paclitaxel treated breast cancer cells when compared to paclitaxel alone treatment. Thus, the present results illustrate the chemosensitizing property of auranofin in MDA-MB-231 and MCF-7 breast cancer cell lines. Citation Format: N. Rajendra Prasad, Deepika Natarajan. Auranofin sensitizes paclitaxel induced apoptotic cell death through oxidative mechanism in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5311.

Comparison of Ziehl Neelsen Stain, Auramine Rhodamine Stain and Culture Sensitivity of AFB in Routine and Concentrated Pleural Fluid

Background: Nearly one third of the global population is infected with mycobacterial tuberculosis. Pleural tuberculosis accounts for 20% of extrapulmonary tuberculosis. The diagnosis of tuberculous pleural effusion is difficult because of the low detection rate of different diagnostic tests like microscopy and culture. Current study aimed to compare the detection rate of different tests in non-concentrated and concentrated pleural fluid.&#x0D; Methods: A hospital based prospective cross sectional study was carried out over one year duration in the Medicine Department of Bir Hospital. A total of 52 cases were enrolled. Detailed history taking and physical examination; radiological, hematological and serum biochemical investigations were performed. Thoracocentesis was performed in all the patients; 20 ml pleural fluid was sent for microscopy with ZN and AR staining as well as for AFB culture. Up to 500ml of pleural fluid was heparinized and kept on cylindrical jar for two hours and 50 ml of sediment was also sent for microscopy and culture within one hour. The results obtained were documented and analysis was done.&#x0D; Results: A total of 52 patients, 31 (59.6%) males and 21 (40.4%) females were included. Their mean age of study participants was 38.67 ± 17.71 (range 16-82 years). Common presentations were fever (94.2%), cough (92.3%), breathlessness (84.6%), chest pain (65.4%) and significant weight loss (59.6%). Haemoptysis was present in 13.5%, anemia in 48.1%, enlarged cervical lymph nodes in 5.8% of the patients. The detection rates of ZN stain, AR stain and AFB culture in non-concentrated pleural fluid were 3.8%, 9.6% and 11.5% respectively. The detection rates for the same tests using concentrated pleural fluid of the same patients were 7.7%, 25% and 17.3% respectively. Differences in detection rate with AR stain and AFB culture for non- concentrated and concentrated pleural fluid were statistically significant (p value &lt;0.01).&#x0D; Conclusion: The detection of AFB using microscopy with ZN and AR staining as well as culture in solid media is low. The detection rate was significantly increased by using concentrated pleural fluid sample for microscopy and culture.

Triple Negative Breast Cancer (TNBC) Cells Growth is Inhibited by High Dose Ascorbic Acid

ObjectiveAccording to the World Health Organization report, the incidence of breast cancer mortality is very high worldwide with 2.1 million women are being affected each year and death are estimated at 627,000 women. There are several subtypes of breast cancer. One of these subtypes is known as triple‐negative breast cancer (TNBC). TNBC doesn’t have the receptors for the hormone estrogen, nor progesterone, nor express HER2/neu; therefore, TNBC doesn’t respond to hormonal nor targeted therapies which makes TNBC difficult to treat.The desired outcome of the cancer therapies is to decrease the proliferative activity of the cancer cells, to minimize drug resistance, and to decrease the toxicity of the anticancer drugs. Ascorbic acid has gained popularity as preventive agent for cancer due to its minimal cytotoxic to normal cellular tissue; however, the use of ascorbic acid for the treatment of cancer has controversial history. Taking oral ascorbic acid had no or little effect on cancer treatment, but recent discovery that intravenous ascorbic acid can provide 100‐fold higher plasma levels than oral intake has renewed interest on the role of ascorbic acid in the cancer treatment.HypothesisAt high dose (pharmacological concentrations), ascorbic acid may act as a prodrug to deliver extracellular H2O2 that is selectively toxic to cancer cells, but non‐toxic and well tolerated to normal cells. It is postulated that TNBC cells growth will be inhibited by high dose of ascorbic acid.MethodsAscorbic acid was buffered to pH 7 with sodium hydroxide and prepared immediately before use. MDA‐MB‐231 cells was used as TNBC model. MTT assay, Hoechst33342, H2DCFDA, and Rhodamine123 staining using fluorescent microscope were conducted to determine cell viability, chromatin inducing apoptosis, intracellular generation of ROS, and mitochondrial membrane potential, respectively. Western Blot to detect different signaling protein expressions such as Poly (ADP‐Ribose) polymerase (PARP), Casp‐3, Casp‐9, P‐53, Bax, Bcl2, Apaf‐1, MMP2, MMP 9, BRCA1, BRCA2, Bim, Map kinases were conducted to test the hypothesis that ascorbic acid was effective to kill TNBC cells.ConclusionThis finding suggests that high dose ascorbic acid has a potential candidate as effective anti‐cancer for TNBC. At 1.6 mM, ascorbic acid killed MDA‐MB‐231, a TNBC cells by causing increased in caspase 3‐9 activities and by decreasing cell cycle regulatory proteins, Mitogen activated kinase 1‐4, and reactive oxygen species.

A Portable Controllable Compressive Stress Device to Monitor Human Breast Cancer Cell Protrusions at Single-Cell Resolution.

In vitro devices offer more numerous methods than in vivo models to investigate how cells respond to pressure stress and quantify those responses. Several in vitro devices have been developed to study the cell response to compression force. However, they are unable to observe morphological changes of cells in real-time. There is also a concern about cell damage during the process of harvesting cells from 3D gels. Here we report a device employing transparent, thin gel layers to clamp cells between the interfaces and applied a controllable compression force by stacking multiple layers on the top. In this approach, cells can be monitored for alteration of cellular protrusions, whose diversity has been proven to promote cancer cell dissemination, with single-cell resolution under compression force. Furthermore, p-Rac-1 and rhodamine staining on the device directly to confirm the actin filaments of lamellipodia. The method was able to fulfill real-time live-cell observation at single-cell resolution and can be readily used for versatile cell analysis. MDA-MB-231 and MCF7 breast cancer cells were utilized to demonstrate the utility of the device, and the results showed that the stimuli of compression force induce MDA-MB-231 and MCF7 to form lamellipodia and bleb protrusions, respectively. We envision the device may be used as a tool to explore mechanisms of membrane protrusion transitions and to screen drug candidates for inhibiting cancer cell protrusion plasticity for cancer therapy.

Detection of Coccidian Parasites using Auramine Rhodamine Stain: A Rapid and Sensitive Tool

Introduction: Intestinal coccidian parasites known to cause opportunistic infections have increased since the past years and are often indistinguishable from other forms of community- acquired diarrhoea, which suggests a need for proper and rapid diagnostic techniques to recover and identify the organism. Aim: To investigate if Auramine Rhodamine (AR) staining is a sensitive and time conserving staining technique in contrast to modified acid fast stain. Materials and Methods: A descriptive observational study was conducted at Nizam’s Institute of Medical Sciences, Hyderabad, India, over a period of three months (i.e., from July to September 2019). A total of 100 stool samples received by the Department of Microbiology were subjected to macroscopic and microscopic examination by saline mount, iodine mount, kinyoun acid fast stain, modified AR staining. Results: In the present study the prevalence of coccidian infections were observed to be 6%. Coccidian parasites reported were Cryptosporidium species and Cystoisospora species immunocompromised patients were found to be more prone to infections with coccidian parasites. The AR stain showed 100% agreement with modified acid fast stain. Conclusion: This study concluded that the AR stain is a rapid and better stain than kinyoun acid-fast stain for the detection of coccidian parasites.

Inorganic titanium dioxide nanoparticles induces cytotoxicity in colon cancer cells

Globally, colorectal cancer (CRC) is responsible for 10% of the commonly diagnosed cancers. Almost ten million colo-rectal cancer deaths occurred in 2020. Existing treatment strategies often led to several off-target and adverse effects. A variety of studies have shown the anti-cancer potential of titanium oxide nanoparticles (TiO2). Therefore, in this study, we have synthesized TiO2 using Lactobacillus and its cytotoxic potential has been evaluated in HT-29 cells, a human CRC cell line. The morphology of TiO2 nanoparticles was analyzed by scanning electron microscope. The cytotoxicity was investigated by MTT assay. Apoptosis-related morphology was analyzed by acridine orange/ethidium bromide staining. Mitochondrial health was analyzed by rhodamine staining. Caspase 3 was analyzed by immunofluorescence. Lactobacillus mediated TiO2 treatments induced cytotoxicity in HT-29 cells. These nanoparticles also induced intracellular reactive oxygen species (ROS) generation and caused apoptosis-related morphological changes in colon cancer cells. TiO2 treatments decreased bcl-2 and increased bax, apaf-1, caspase-9 and −3 gene expressions and this treatment also caused mitochondrial dissipation and cytochrome c release. The current study results suggest that TiO2 is capable of inducing cytotoxicity in colon cancer cells by intracellular ROS accumulation. The ROS induction was associated with mitochondrial toxicity and cytochrome c increase can be related to the induction of intrinsic apoptosis. These results indicate that TiO2 could cause apoptosis by activating intrinsic apoptotic pathway and generating ROS in HT-29 cells. These results suggest that TiO2 can be used as a lung cancer therapeutic agent after necessary in vivo and clinical studies.

Arbutin exerts anticancer activity against rat C6 glioma cells by inducing apoptosis and inhibiting the inflammatory markers and P13/Akt/mTOR cascade.

Gliomas are a type of brain cancer that occurs in the supporting glial cells of the brain. It is highly malignant and accountsfor 80% of brain tumors with high mortality and morbidity. Phytomedicines are potent alternatives for allopathic drugs which cause side effects. They have beenused from ancient times by traditional Chinese, Ayurveda, and Siddha medicine. Arubtin is a glycoside phytochemical extracted from plants and belongs to the family of Ericaceae. Arbutin possesses various pharmacological properties such as anti-inflammatory, antioxidant, antitumor, and so on. Hence in the present study, we analyzed the anticancer potency of arbutin against rat C6 glioma cells. Rat C6 glioma cells were procured from American Type Culture Collection and the cells were cultured in Roswell Park Memorial Institute-1640 medium. To assess the cytotoxicity effect of the arbutin against C6 glioma cells, an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test was performed with different doses from 10 to 60 µM. Arbutin effectively induced apoptosis in the cells and the IC50 dose was obtained at 30 µM. For further studies, we selected the 30 µM IC50 dose and a higher dose of 40 µM. Reactive oxygen species (ROS) generated were analyzed with DCFDA/H2DCFDA stain and the destruction of mitochondrial membrane permeability which is the initiator of apoptosis was analyzed with a cationic stain Rhodamine 123. Dual staining with acridine orange and ethidium bromide was performed to assess the viable and dead cells. Cell adhesion propertiesof glioma cells were analyzed with Matrigel assay. The apoptotic, inflammatory, and phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling molecules were analyzed with quantitative polymerase chain reaction (qPCR) analysis to confirm the anticancer effect of arbutin. Arbutin generated excessive ROS and disrupted the mitochondrial membrane, which induced apoptosis in cells, it also inhibited the cell adhesion property of C6 glioma cells. qPCR analysis clearly indicates arbutin increases the apoptotic genes and decreased the inflammatory and PI3K/mTOR signaling molecules. Overall, our results authentically confirm that arbutin can be a potent alternative for treating glioma.