Acridine Orange/propidium Iodide Research Articles

Green Hydrothermal Synthesis of Gallic Acid Carbon Dots: Characterization and Cytotoxic Effects on Colorectal Cancer Cell Line.

Colorectal cancer remains a leading cause of cancer-related deaths worldwide, necessitating the development of novel therapeutic approaches. Carbon dots (CDs) have emerged as promising nanoparticles for biomedical applications due to their unique properties. Gallic acid (GA), a known anticancer agent, is effective against various tumor types. This study explores the potential of gallic acid-derived carbon dots (GA-CDs) as an innovative anticancer agent against HCT-116 colorectal cancer cells, focusing on apoptosis signaling pathways. GA-CDs were synthesized using a one-pot hydrothermal method. Characterization was conducted using transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, and ultraviolet-visible (UV-Vis) absorption spectroscopy. The cytotoxicity of GA and GA-CDs on HCT-116 cells was evaluated using the MTT assay at various concentrations over 24 and 48 hours. Cellular uptake was assessed via fluorescence microscopy, and apoptosis was analyzed using acridine orange/propidium iodide (AO/PI) staining. Total RNA extraction followed by complementary DNA (cDNA) synthesis via RT-PCR was performed, and real time-PCR (Q-PCR) was conducted to examine the expression of apoptosis-related genes Caspase-3, Bax, and Bcl-2. Characterization confirmed the successful synthesis of spherical GA-CDs. GA-CDs exhibited dose- and time-dependent cytotoxicity, with IC50 values of 88.55 µg/mL for GA-CDs and 192.2 µg/mL for GA after 24 hours. Fluorescence microscopy confirmed the efficient uptake of GA-CDs by HCT-116 cells. AO/PI staining showed a significant increase in apoptotic cell numbers after treatment with GA-CDs. Q-PCR analysis revealed overexpression of Caspase-3 and Bax genes in GA-CD-treated cells, though no significant changes were observed in the expression of Bcl-2 or the Bax/Bcl-2 ratio. GA-CDs demonstrated potent anticancer properties by inducing apoptosis and reducing cell viability in HCT-116 cells. These findings suggest the potential of GA-CDs as a novel therapeutic agent for colorectal cancer treatment, warranting further investigation into their mechanism of action and in vivo efficacy.

Characterizing the health of transduced CAR-T cells using high-throughput image cytometry

Abstract Chimeric antigen receptor (CAR)-T cell therapy is one of the novel cellular therapeutic options for cancer patients. In this work, we developed a rapid cell viability and apoptosis detection method to characterize the cell health and recovery of SubT1 cells after the plasmid-based electroporation process to create CAR expression cells using the Cellaca™ PLX Image Cytometer. Viability measurement of transduced SupT1 cells was performed by staining with acridine orange/propidium iodide (AO/PI). In addition, early and late-stage apoptosis were detected by staining transduced SupT1 cells with Annexin V/PI and Caspase3/RubyDead, respectively. Results showed that the introduction of plasmids, other than the electroporation process itself, may induce 20 – 30% additional cell death in the process, indicated by viability results measured on Day 1 post transduction. Among all the Annexin V or Caspase3 positive transduced SupT1 cells on Day 1 (22 – 34%), 70 - 80% of these cells were double positive in both Annexin V and PI, or both Caspase3 and RubyDead, and only 20 – 30% of them were in the early-stage/late-stage apoptosis. Subsequently, the SupT1 cells were quickly recovered with their viabilities increased to >90% on Day 3. With such capability of rapid detection of cell viability and apoptosis, Cellaca™ PLX Image Cytometer can potentially support the characterization of factors that affects the quality of CAR-T cells in the manufacturing process.

تقييم نشاط التئام الجروح وتحفيز موت الخلايا المبرمج لمشتقات الكينازولينون الجديدة

Background: Chemotherapeutic medication treatment for cancer is typically used in conjunction with other techniques as part of a routine regimen. It is well established that the capacity of different chemotherapeutic drugs to induce apoptosis is correlated with their anticancer efficacy. Quinazolinone-based drugs have demonstrated excellent responses from several cancer cell types. These substances have a lot of potential for use as building blocks in the creation of apoptosis inducers. Objective: To assess the new quinazolinone derivatives (M1 and M2) that were recently synthesized for their potential to halt wound healing and to use the acridine orange/propidium iodide (AO/PI) double stain to assess their capacity to induce apoptosis in the chosen cancer cell lines. Methods: Using the breast carcinoma cell line (MCF-7) and the lung adenocarcinoma cell line (A549), two quinazolinone derivatives (M1 and M2) were investigated for their capacity to inhibit wound healing and induce apoptosis. Results: In both cell lines, the chemicals were found to be effective inducers of apoptosis and to considerably limit wound healing. Conclusions: In cancer cell lines (MCF-7 and A549), compounds M1 and M2 efficiently inhibited wound repair and triggered apoptosis.

Signalling Mechanism in TRPM2-dependent Copper- induced HT22 Cell Death

Copper (Cu) is one of the critical elements needed by the human body. However, this metal can cause cytotoxicity when present in excess amounts. In this study, we used HT22 hippocampus cells to examine the function of the Ca2+-permeable transient receptor melastatin 2 (TRPM2) channel in Cu-induced neuronal cell death and the underlying mechanisms. Immunocytochemistry, single-cell imaging, acridine orange/propidium iodide (AO/PI) cell death assay and immunofluorescence microscopy were applied to interpret the mechanisms involved in Cu-induced HT22 cell death. Treatment of 30-300 μM Cu induced an increase in the [Ca2+]i in HT22 cells. Further analysis indicates that Cu exposure induced substantial HT22 cell death. Such response on HT22 cells was significantly inhibited by 2-aminoethoxydiphenyl borate (2-APB) and N-(p-amylcinnamoyl)anthranilic acid (ACA), TRPM2 channel inhibitors. Furthermore, Cu-induced HT22 cell death was suppressed by pharmacologically inhibiting poly(ADPR) polymerase (PARP) using PJ-34 and DPQ. It is known that the activation of TRPM2 channel is via the increase of intracellular reactive oxygen species (ROS). A significant concentration-dependent increase in the generation of ROS was observed in HT22 following the treatment with 30-300 μM Cu. Additionally, Cu-induced HT22 cell death was ablated by inhibiting PKC using CTC, a PKC inhibitor and NADPH oxidase (NOX) using DPI, a NOX generic inhibitor and GKT137831, a NOX1/4-specific inhibitor. Overall, our present study provides evidence suggesting that PKC/NOX/ROS/PARP is an important signalling pathway in Cu-induced activation of the TRPM2 channel and increase in the [Ca2+]i which eventually results in the toxicity of HT22 cells. These results provide new insights into the mechanisms of neurological-associated Cu-induced diseases.

Effect of Black Rice Bran ‘Sembada Hitam’ on T47D Breast Cancer Cells

Indonesians have a rich tradition of incorporating black rice into their diet as a functional food due to its high antioxidant content. This research investigates the effects of the ethanolic extract of "Sembada Hitam" black rice bran on T47D breast cancer cells, specifically its cytotoxic, cell growth, and apoptotic induction properties. The MTT assay method was used to evaluate the cytotoxicity of the black rice bran extract on T47D cells after 24 and 48 hours of incubation. The acridine orange/propidium iodide (AO/PI) dye double staining method was employed to assess the apoptosis-inducing properties of the extract. Additionally, a cell growth assay was conducted to evaluate the effect of the extract on cell growth, with observations recorded on days 0, 3, and 6. The results revealed that the ethanolic extract of black rice bran "Sembada Hitam," when administered at various concentrations ranging from 7.81 to 1000 µg/mL, did not exhibit cytotoxic effects on T47D cells during the 24 and 48 hours of incubation. The cell growth assay revealed that T47D cells treated with the ethanolic extract of "Sembada Hitam" black rice bran at concentrations of 250 and 500 µg/mL exhibited lower growth rates than the 1% DMSO group from the 3rd to 6th day after incubation. The ethanolic extract of "Sembada Hitam" at concentrations of 250, 500, and 1000 µg/mL resulted in cell death of 10.64 ± 2.98%, 9.99 ± 5.87%, and 5.84 ± 0.78%, respectively. In conclusion, this study found that the ethanolic extract of "Sembada Hitam" black rice bran did not demonstrate significant cytotoxic effects on T47D breast cancer cells and is, therefore, unlikely to be a suitable candidate for an anti-cancer agent.

Evaluation of cytotoxic activity against A549 human lung cancer cells using green synthesized N-Cholyl D-Penicillamine encapsulated silver and gold nanoparticles

Synthesis of noble metal nanoparticles (MNPs) using a biocompatible template provides a biologically safe, cost effective, and environment friendly approach in view of biomedical applications. In this manusctipt, synthesis of gold and silver nanoparticles (Au and Ag NPs) using N-cholyl D-penicilamine (NCPA) as an effective reducing and stabilizing agent at below the critical micellar concerntration (CMC) under sunlight exposure has been reported. The formation of NPs were optimized by changing the concentration of NCPA and pH of the medium. The synthesized Au and Ag NPs were characterized by UV-visible, FT-IR, DLS, Zeta potential and HR-TEM techniques. The average size of Au and Ag NPs were found to be 10.3 ± 0.4 and 9.1 ± 0.6 nm, respectively and exhibit stability for more than six month. The prepared NPs showed low toxicity on human erythrocytes (RBCs) even at their higher concentrations (120 μg/mL). The anticancer activity of NCPA capped NPs on A549 cells were investigated by cell viability assay, apoptosis analysis using MTT assay and Acridine Orange/Propidium Iodide (AO/PI) dual fluorescent staining asay. The results obtained from these studies suggest that the synthesized NPs showed potent cytotoxicity on A549 cells in a dose-dependent manner. Further, AgNPs showed better cytotoxicity as compared to AuNPs.

Natural Gallic Acid and Methyl Gallate Induces Apoptosis in Hela Cells through Regulation of Intrinsic and Extrinsic Protein Expression.

Induction of apoptosis is one of the targeted approaches in cancer therapies. As previously reported, natural products can induce apoptosis in in vitro cancer treatments. However, the underlying mechanisms of cancer cell death are poorly understood. The present study aimed to elucidate cell death mechanisms of gallic acid (GA) and methyl gallate (MG) from Quercus infectoria toward human cervical cancer cell lines (HeLa). The antiproliferative activity of GA and MG was characterised by an inhibitory concentration using 50% cell populations (IC50) by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Cervical cancer cells, HeLa, were treated with GA and MG for 72 h and calculated for IC50 values. The IC50 concentration of both compounds was used to elucidate the apoptotic mechanism using acridine orange/propidium iodide (AO/PI) staining, cell cycle analysis, the Annexin-V FITC dual staining assay, apoptotic proteins expressions (p53, Bax and Bcl-2) and caspase activation analysis. GA and MG inhibited the growth of HeLa cells with an IC50 value of 10.00 ± 0.67 µg/mL and 11.00 ± 0.58 µg/mL, respectively. AO/PI staining revealed incremental apoptotic cells. Cell cycle analysis revealed an accumulation of cells at the sub-G1 phase. The Annexin-V FITC assay showed that cell populations shifted from the viable to apoptotic quadrant. Moreover, p53 and Bax were upregulated, whereas Bcl-2 was markedly downregulated. Activation of caspase 8 and 9 showed an ultimate apoptotic event in HeLa cells treated with GA and MG. In conclusion, GA and MG significantly inhibited HeLa cell growth through apoptosis induction by the activation of the cell death mechanism via extrinsic and extrinsic pathways.

Characterization and comparison of acridine orange/propidium iodide and acridine/DAPI viability detection methods for cell and gene therapy development

Abstract Cell viability is one of the critical quality attributes (CQAs) for analyzing and characterizing cellular therapeutic products. The characterization of cell viability is essential to ensure quality, safety and efficacy during cell and gene therapy development. In this work, we characterized and compared two commonly used viability dual-staining methods, acridine orange/propidium iodide (AO/PI) and acridine orange/4′,6-diamidino-2-phenylindole (AO/DAPI) using the Cellaca MX high-throughput cell counter. In general, the AO/DAPI method provided higher viability results than AO/PI, with the measured viability difference between these two methods ranging from 0% to 24%. To identify the sources of the measured viability difference, we tested a list of factors including cell dying process, staining time and initial viability levels. First, we observed a larger viability difference from naturally-dying Jurkat cells compared to the result from heat-killed cells, suggesting that the differences may depend on the cell dying process. Second, we found that AO/PI provided more consistent cell count and viability than AO/DAPI during the first 30 min of staining time. Finally, we showed that measured viability difference might be larger for low-viability, naturally-dying Jurkat cells, as compared to healthy samples. The results from our characterization studies may benefit the selection of viability methods that are fit-for-purpose for analyzing cellular therapeutic products.

Abstract 5332: Characterization and comparison of acridine orange/propidium iodide and acridine/DAPI viability detection methods for cell and gene therapy development

Abstract Cell viability is one of the critical quality attributes (CQAs) for cell and gene therapy products. Characterization of cell viability, in addition to cell identity, cell count and other CQAs, are essential to ensure the quality, safety and efficacy throughout various developmental stages of cell and gene therapies. To determine cell viability, a variety of viability detection methods have been used, such as trypan blue (TB) exclusion assay, and fluorescence-based dyes including acridine orange/propidium iodide (AO/PI), acridine orange/4′,6-diamidino-2-phenylindole (AO/DAPI), all of which have been employed on flow or image-based cytometry systems. In this work, we employed the Cellaca MX high-throughput cell counter to characterize and compare AO/PI and AO/DAPI viability detection methods. First, we measured the viabilities of different ratios of viable and non-viable Jurkat cells created via two cell dying processes. Next, viabilities of the stained cells were measured multiple times within a 30 min staining time for each method in order to monitor the trend of viability changes. The viability results showed that the AO/DAPI method were generally higher than AO/PI method with differences ranging from 0% to 24%. We also observed the discrepancy of the measured viability differences between naturally-dying and heat-killed Jurkat cells, suggesting that the viability difference might be dependent on the cell dying process. In the time-dependent viability characterization, results from AO/DAPI method showed increasing dead cell counts leading to decreasing viability, revealing an on-going staining process. In contrast, results from AO/PI method showed more consistent cell count and viability measurements, indicating a more instantaneous staining process. Lastly, we conducted a cell proliferation assay as an orthogonal method to confirm replicability of the low viability cell samples. The proliferation assay results showed no significant increase in cell concentrations within the 4-day time frame. The viability results before the culture were approximately 0% for both naturally-dying and heat-killed Jurkat cells, using the AO/PI staining method, and 12% and 9% using the AO/DAPI respectively. Furthermore, spiked samples of natural-dying and heat-killed cells with healthy cells (>90% viability) were generated and measured, which showed exponential growth. The viability results of these spiked samples before the culture were 11% and 9% by AO/PI and 17% and 18% by AO/DAPI, respectively. Taken together, these results suggest that cell viability methods need to be optimized for different fluorescent stains in order to generate reliable, robust, and fit-for-purpose results for the downstream assays for cell and gene therapy products.. Citation Format: Yongyang Huang, Samir Patel, Mackenzie Pierce, Carolina Franco Nitta, Bo Lin, Leo Chan. Characterization and comparison of acridine orange/propidium iodide and acridine/DAPI viability detection methods for cell and gene therapy development. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5332.

Three-dimensional, label-free cell viability measurements in tissue engineering scaffolds using optical coherence tomography.

In the field of tissue engineering, 3D scaffolds and cells are often combined to yield constructs that are used as therapeutics to repair or restore tissue function in patients. Viable cells are often required to achieve the intended mechanism of action for the therapy, where the live cells may build new tissue or may release factors that induce tissue regeneration. Thus, there is a need to reliably measure cell viability in 3D scaffolds as a quality attribute of a tissue-engineered medical product. Here, we developed a noninvasive, label-free, 3D optical coherence tomography (OCT) method to rapidly (2.5min) image large sample volumes (1 mm3 ) to assess cell viability and distribution within scaffolds. OCT imaging was assessed using a model scaffold-cell system consisting of a polysaccharide-based hydrogel seeded with human Jurkat cells. Four test systems were used: hydrogel seeded with live cells, hydrogel seeded with heat-shocked or fixed dead cells and hydrogel without any cells. Time series OCT images demonstrated changes in the time-dependent speckle patterns due to refractive index (RI) variations within live cells that were not observed for pure hydrogel samples or hydrogels with dead cells. The changes in speckle patterns were used to generate live-cell contrast by image subtraction. In this way, objects with large changes in RI were binned as live cells. Using this approach, on average, OCT imaging measurements counted 326 ± 52 live cells per 0.288 mm3 for hydrogels that were seeded with 288 live cells (as determined by the acridine orange-propidium iodide cell counting method prior to seeding cells in gels). Considering the substantial uncertainties in fabricating the scaffold-cell constructs, such as the error from pipetting and counting cells, a 13% difference in the live-cell count is reasonable. Additionally, the 3D distribution of live cells was mapped within a hydrogel scaffold to assess the uniformity of their distribution across the volume. Our results demonstrate a real-time, noninvasive method to rapidly assess the spatial distribution of live cells within a 3D scaffold that could be useful for assessing tissue-engineered medical products.

Nitrogen and copper-doped saffron-based carbon dots: Synthesis, characterization, and cytotoxic effects on human colorectal cancer cells

AimDoped carbon dots (CDs) have attracted tremendous attention in cancer therapy. We aimed to synthesize copper, nitrogen-doped carbon dots (Cu, N-CDs) from saffron and investigated their effects on HCT-116 and HT-29 colorectal cancer (CRC) cells. Main methodsCDs were synthesized by hydrothermal method and characterized by transmission electron microscopy (TEM), energy-dispersive X-ray (EDX), Fourier transform infrared (FT-IR) spectroscopy, ultraviolet-visible (UV–Vis) absorption spectroscopy, and fluorescence spectroscopy. HCT-116 and HT-29 cells were incubated with saffron, N-CDs, and Cu, N-CDs for 24 and 48 h for cell viability. Cellular uptake and intracellular reactive oxygen species (ROS) were evaluated by immunofluorescence microscopy. Oil Red O staining was used to monitor lipid accumulation. Apoptosis was evaluated using acridine orange/propidium iodide (AO/PI) staining and quantitative real-time polymerase chain reaction (Q-PCR) assay. The expression of miRNA-182 and miRNA-21 was measured by Q-PCR, while the generation of nitric oxide (NO) and lysyl oxidase (LOX) activity was calculated by colorimetric methods. Key findingsCDs were successfully prepared and characterized. Cell viability decreased in the treated cells dose- and time-dependently. HCT-116 and HT-29 cells uptook Cu, N-CDs with a high level of ROS generation. The Oil Red O staining showed lipid accumulation. Concomitant with an up-regulation of apoptotic genes (p < 0.05), AO/PI staining showed increased apoptosis in the treated cells. In comparison to control cells, NO generation, and miRNA-182 and miRNA-21 expression significantly changed in the Cu, N-CDs treated cells (p < 0.05). SignificanceThe results indicated that Cu, N-CDs could inhibit CRC cells through the induction of ROS generation and apoptosis.

Olea europaea Leaf Phenolics Oleuropein, Hydroxytyrosol, Tyrosol, and Rutin Induce Apoptosis and Additionally Affect Temozolomide against Glioblastoma: In Particular, Oleuropein Inhibits Spheroid Growth by Attenuating Stem-like Cell Phenotype.

The effects of Olea europaea leaf extract (OLE) phenolics, including oleuropein (OL), hydroxytyrosol (HT), tyrosol (TYR), and rutin against glioblastoma (GB), independently and in combination with temozolomide (TMZ), were investigated in T98G and A172 cells. Cell growth was assessed by WST-1, real-time cell analysis, colony formation, and cell cycle distribution assays. A dual acridine orange propidium iodide (AO/PI) staining and annexin V assay determined cell viability. A sphere-forming assay, an intracellular oxidative stress assay, and the RNA expression of CD133 and OCT4 investigated the GB stem-like cell (GSC) phenotype. A scratch wound-healing assay evaluated migration capacity. OL was as effective as OLE in terms of apoptosis promotion (p < 0.001) and GSC inhibition (p < 0.001). HT inhibited cell viability, GSC phenotype, and migration rate (p < 0.001), but its anti-GB effect was less than the total effect of OLE alone. Rutin decreased reactive oxygen species production and inhibited colony formation and cell migration (p < 0.001). TYR demonstrated the least effect. The additive effects of OL, HT, TYR and rutin with TMZ were significant (p < 0.001). Our data suggest that OL may represent a novel therapeutic approach against GB cells, while HT and rutin show promise in increasing the efficacy of TMZ therapy.

Synthesis and Evaluation of New Derivatives of Busulfan as an Anti-carcinogenic Drug against k562 Cancer Cells Using the AO / PI Method

Background: Busulfan is a DNA alkylating drug used to treat chronic myeloid leukemia (CML). This type of leukemia affects bone marrow cells or myocytes, which make up bone marrow tissue having a chronic process. Leukemia is a progressive and malignant disease of the body's hematopoietic organs. The disease is caused by the underdevelopment and proliferation of white blood cells and their precursors in the blood and bone marrow. Objective: The synthesis of Busulfan drug and the conjugation of Busulfan from the anomeric position of acetylated galactose as a new derivative of Busulfan and its anti-cancer effect on K562 cancer cells are among the most important objectives of this study. Methods: In this work, galactose was acetylated with acetic anhydride in the presence of sodium acetate, then the anomeric position of acetylated carbohydrate deacetylated using imidazole to obtain the final product through the reaction of Busulfan. Anti-cancer activity of the product was evaluated against K562 cancer cells. The proliferation and survival of K562 cancer cells and PBMCs (peripheral blood mononuclear cells) using the AO/PI method were studied. Furthermore, 1- (2, 3, 4, 6-tetra-O-acetyl-Iβ-D-galactopyranosyl) -4- (methyl sulfonyl oxy) butane was evaluated against cancer cells as a new derivative. Results: In this study, the Acridine Orange / Propidium Iodide (AO/PI) method was performed using fluorescence microscopy to evaluate the efficacy of a synthesized product. The effective dose of 0.02 mg/L of 1- (2, 3, 4, 6-tetra-O-acetyl-Iβ-D-galactopyranosyl) -4- (methyl sulfonyl oxy) butane was observed to cause 96% of cancer cells necrosis. Conclusion: 1- (2, 3, 4, 6-tetra-O-acetyl-Iβ-D-galactopyranosyl) -4- (methyl sulfonyl oxy) butane as a new derivative of Busulfan acts non-specifically in the cell cycle by inhibiting DNA activity by alkylation and binding to the DNA strand. Alkylating agents usually bind an alkyl group to the number seven nitrogen atom in the guanine nucleotide in a DNA molecule.

Anti-cancer effects of PLGA nanoparticles loaded with anbarnesa smoke organic extract on A2780 ovarian cancer cell line

ABSTRACT There are many traditional agents whose combination with new technologies can offer effective therapeutic impacts. Accordingly, the purpose of this study was to evaluate the effects of PLGA nanoparticles (NPs) loaded with anbarnesa smoke organic extract (P-ASOE) against A2780 ovarian cancer cell line. Thus, nanoparticles were synthesised using W1/O/W2 method and characterised by FESEM, DLS and FTIR techniques. Cytotoxicity of P-ASOE on A2780 and healthy (HFF) cells was assessed using MTT assay. Acridine Orange/Propidium Iodide (AO/PI) staining, flow cytometry and expression profiles of P53, TNFα, CAS-3 and CAS-9 genes were recruited to evaluate the effect of P-ASOE on apoptosis of A2780 cells. The antiangiogenic effect of P-ASOE was studied through chorioallantoic membrane (CAM) assay as well as evaluation of VEGF and VEGFR gene expression. The histological effects of P-ASOE on healthy and cancerous tissues of mouse models were investigated. The results showed that the synthesised NPs had dose- and time-dependent cytotoxic effects on A2780 cells but not on HFF cells. In addition, all assays indicated pro-apoptotic effects of P-ASOE, which significantly reduced number and length of blood vessels as well as length and weight of chick embryos. The expression levels of VEGF and VEGFR genes were also significantly decreased under the influence of NPs. According to in vivo examinations, P-ASOE treatment reduced tumour size while having no significant histological effect on healthy tissues. All findings were in favour of the anti-cancer potential of P-ASOE against A2780 cancer cell line.

Cytotoxic Activity of Aquilaria malaccensis Ethanolic Leaves Extract on Human Chronic Myeloid Leukaemia K-562 Cell Line

Aquilaria malaccensis, also known as “Pokok Karas” in Malaysia, has various benefits and widely used in Chinese herbal medicines. Natural products such as medicinal plant have been found to be effective anti-cancer agents, killing cancer cells with fewer side effects than chemotherapy. Therefore, the purpose of this study was to evaluate the cytotoxic activities of ethanolic extract of A. malaccensis on the human chronic myeloid leukaemia K-562 cell line. In this study, cytotoxic activities of ethanolic extract of A. malaccensis leaves on K-562 cells was determined by using (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) (MTT) assay. K-562 cells were treated with 70% ethanolic extracts and incubated for 24, 48 and 72 hours. The half-maximal inhibitory concentration (IC50) of ethanolic extract of A. malaccensis leaves was observed at 72 hours of incubation; 90 µg/mL for 70% ethanolic extract. Moreover, the morphological changes of K-562 cells were observed by using acridine orange/ propidium iodide (AO/PI) dual staining assay. The results showed that the ethanolic extract of A. malaccensis leaves induced K-562 cells into apoptotic cell death mode manner. This finding suggested that the ethanolic extract of A. malaccensis has the potential to become an anti-cancer agent for chronic myeloid leukaemia K-562 cell line.

Cytotoxic activity of hantap (Sterculia oblongata Mast) leaves extract against breast cancer cells line (MCF7/HER2): the effect on the expression of HER2 mRNA and the apoptosis

Hantap leaves (Sterculia oblongata Mast) has been used traditionally to treat breast cancer in Palu, Central Sulawesi. However, its use is just based on empirical evidences rather than scientific evidences. The study aimed to investigate the cytotoxic activity of hantap leaves extracts against breast cancer cells line. The effect of this extract on the HER2 expression and the apoptosis was also evaluated. The hantap (S. oblongata Mast) leaves extracts were prepared by consecutive maceration method using n-hexane, methanol and water, respectively. The cytotoxic activity against MCF7/HER2 breast cancer cells line was evaluated using the MTT assay with doxorubicin as a positive control. The HER2 mRNA expression was examined using RT-PCR and the apoptosis after 24 h incubation was examined using a fluorescence microscope after AO-PI (acridine orange-propidium iodide) staining. Among three extracts tested, the methanolic extract exhibited the most cytotoxic against MCF7/HER2 cells with an IC50 of 91.25 μg/mL. Therefore, the methanolic extract was subjected to further study. The methanolic extract at concentration of 1/2IC50; 1IC50 and 2IC50 μM induced 6.8; 26.3 and 25.3% apoptosis of the MFC7/HER2 cell lines, respectively. The methanolic extract at concentration of 1/2IC50; 1IC50 and 2IC50 μM inhibited HER2 mRNA expression to be 0.6; 0.25 and 0.33 compared to control cells. In conclusion, the methanolic extract of hantap leaves (S. oblongata Mast) has cytotoxic activity against MCF7/HER2 breast cancer cell lines by induce cells apoptosis and inhibit HER2 mRNA expression. Further study, will be conducted to isolate active constituents as anticancer.

Induction of apoptosis by Eleutherine bulbosa (Mill.) Urb. bulb extracted under optimised extraction condition on human retinoblastoma cancer cells (WERI-Rb-1)

Ethnopharmacological relevanceThe bulb of Eleutherine bulbosa (Mill.) Urb. is an indigenous medicinal plant traditionally used among Dayak people for the management of diabetes, breast cancer, hypertension, stroke, and fertility problems in women. The bulb has been reported with a potent cytotoxic potential but with limited underlying mechanisms. Aim of the studyThis study aimed to investigate the cytotoxic properties of E. bulbosa ethanolic bulb extracted under optimised extraction condition on retinoblastoma cancer cells (WERI-Rb-1) through in vitro cell culture bioassays. The optimised extraction condition has been determined in the previous reports. Materials and methodsCytotoxic assay was analysed through MTT assay. Comparison between non-optimised and optimised extraction condition from E. bulbosa ethanolic bulb extract was evaluated. Morphological assessment of apoptotic cells was conducted through acridine orange propidium iodide (AOPI) staining using fluorescence microscopy. Apoptosis assay was carried out through Annexin V-FITC and cell cycle analysis through PI staining. The effect of varying concentrations (IC25, IC50, IC75) of the optimised E. bulbosa ethanolic bulb extract was observed. The mRNA expression was also conducted to confirm the underlying mechanism. ResultsThe optimised E. bulbosa ethanolic bulb extract markedly suppressed the proliferation of retinoblastoma cancer cells significantly with an IC50 value of 15.7 μg/mL as compared to non-optimised extract (p < 0.01). Fluorescence microscopy revealed that retinoblastoma cancer cells manifested early features of apoptosis-like membrane blebbing, chromatin condensation and formation of apoptotic bodies in a dose-dependent manner. The number of apoptotic cells were greatly observed in early and late apoptosis through Annexin V-FITC and the extract also induced cell arrestment as compared to the untreated group. The apoptosis was confirmed with the upregulation of Bax, Bad, p53, Caspase 3, Caspase 8, and Caspase 9 genes meanwhile, Bcl-2, BcL-xL, Nrf-2, and HO-1 genes were downregulated. ConclusionThe optimised E. bulbosa ethanolic bulb extract induced a significant cell death and cell cycle arrestment on retinoblastoma cancer cells. It could be suggested that the induction of apoptosis in retinoblastoma cancer cells may be due to the synergistic effect of the bioactive compounds extracted under optimised extraction condition. Our findings indicated that E. bulbosa bulb could be promising chemotherapeutic potential to treat retinoblastoma cancer cells.

Anti-acanthamoebic activity of methanolic extract of Piper sarmentosum leaves

Aims: Piper sarmentosum or locally known as Kaduk, is a tropical herb plant that was investigated for its phenolic content by previous researchers. The present study aimed at the analysis of crude methanolic extract of P. sarmentosum leaves for phenolic compounds identification and its anti-amoebic properties against pathogenic Acanthamoeba castellanii. Methodology and results: Folin-Ciocalteu assay was used to determine P. sarmentosum leaves methanolic extract (PSLME)’s total phenolic content (TPC). The extract was further characterized by using gas chromatography-mass spectrometry (GC-MS), reverse phase-high performance liquid chromatography (RP-HPLC) and liquid chromatography mass spectrometry (LC-MS) analyses to determine the chemical constituents in methanolic PSLME extract. The cytotoxicity of the extract was evaluated through the determination of inhibition concentration for half of cell population (IC50) of pathogenic A. castellanii followed by cell morphological analysis using inverted light and scanning electron microscopies. Acridine-orange/Propidium iodide (AOPI) staining was also conducted to determine the integrity of cell membrane for quantitative analysis. The results demonstrated that the TPC from PSLME was 142.72 mg [GAE]/g with a total of 33 phenolic compounds identified. The IC50 value obtained for A. castellanii was low (74.64 μg/mL) which indicates promising anti-acanthamoebic activity. Microscopy analyses showed that the plant extract caused cells encystment, in which exhibited by distinctive morphological changes on the cells shape and organelle, as well as shortening of acanthopodia. The dual staining and its quantitative analysis prove compromised membrane integrity in the treated amoeba. Conclusion, significance and impact of study: This finding provides the evidence that PSLME contains active phenolic compounds contributing to the anti-acanthamoebic activity on pathogenic Acanthamoeba species.

Apoptosis Induction with Combined Use of Cisplatin and Fisetin in Cisplatin- Resistant Ovarian Cancer Cells (A2780)

Ovarian cancer is the leading cause of death caused by genital cancers. One of the most common treatments for this type of cancer is chemotherapy by cisplatin, which induces apoptosis in cancer cells. Apoptosis is a type of physiological cell death. Cisplatin chemotherapy usually has several side effects and cellular resistance to cisplatin is a common incidence. In order to overcome these problems, the use of combination therapies using natural substances has been considered. Fisetin is a flavonoid with anti-cancer activity which induces apoptosis. In this study, the apoptosis induced by cisplatin along with Fisetin in cisplatin-resistant ovarian cancer cell line (A2780) was investigated. In the present experimental study, the effect of combined use of Fisetin and cisplatin on ovarian cancer cell lines (A2780) was investigated by using MTT assay. Cell death was also determined by DAPI, acridine orange/propidium iodide, and Annexin/PI assay. Apoptotic gene expression of Bax, BCL-2, caspase 3, and caspase 9 was also assessed by real time PCR. The results of MTT assay indicated that the combined treatment of Fisetin and cisplatin effectively inhibits proliferation of A2780 cells. The results of DAPI staining showed that fragmentation of chromatin in cells occurred in the combined treatment. Acridine orange-propidium iodide staining and Annexin/PI staining showed an increase in the rate of apoptotic cells in cells under combined treatment. The results of the study regarding changes in gene expression also indicated that Bax pro-apoptotic gene expression and BCL-2 anti-apoptotic gene expression increased in cells under treatment; moreover, gene expression of caspases 3 and 9 significantly increased as well. According to the findings of this study, the combined use of cisplatin and Fisetin increases the induction of apoptosis in cisplatin-resistant ovarian cancer cells (A2780); therefore, the combined use of cisplatin and Fisetin can be considered a promising strategy in the treatment of ovarian cancer.

Aktivitas Sitotoksik dan Antiproliferasi Fraksi n-Heksan Biji Al-pukat (Percea americana Mill.) Terhadap sel T47D

Avocado plants are widely used as traditional medicine in Indonesia. This research aimed to determine the cytotoxic and antiproliferation activity of avocado seed extract and fractions on the T47D breast cancer cell line. Extraction was done by maceration method using 96% ethanol, then fractionation was done by liquid-liquid partition using n-hexane, chloroform, and ethyl acetate. Cytotoxic and antiproliferation tests were done by the MTT method. Apoptosis activity was investigated by the double staining method, using acridine orange-propidium iodide as a staining reagent. Results showed that the n-hexane fraction of avocado seed had a cytotoxic activity with IC50 27.9 µg/mL. N-hexane fraction of avocado seed inhibited proliferation and induced apoptosis of T47D cell line.