Synthesis,biological evaluation and molecular docking study of 1-amino-2-aroylnaphthalenes against prostate cancer

A series of functionalized naphthalene was synthesized and screened against human prostate cancer cell line (PC-3). The in vitro antiproliferative activity of the synthesized compounds was evaluated by monitoring their cytotoxic effects against PC-3 cells by using MTT assay. We observed that compound 5f resulted in more than 50% cell death at 14?µM. Treatment of PC-3 cells with 5f provides apoptosis by flow cytometry. Western blotting showed decreased expression of pro-caspase 8 and 9. Our study shows that cancer cell treated with 5f has higher concentration of reactive oxygen species as compare to untreated sample, which facilitate cancerous cell to enter apoptosis. Exact mechanism by which ROS is generated after 5f treatment is still under study. Molecular docking study further strengthens the results obtained from in vitro experiments. Compound 5f can be considered as a promising leads for anticancer agent against prostate cancer cells due to its potent cytotoxic activity and apoptotic effect.     

In vitro apoptotic effect of cassaine-type diterpene amides from Erythrophleum fordii on PC-3 prostate cancer cells

Cytotoxic activity-guided fractionation of Erythrophleum fordii led to the isolation of two new cassaine diterpenoid–diterpenoid amide dimers, erythrophlesins H–I (1, 2). Spectral data indicated that they consist of asymmetrical dimeric structure via an ester bond between two cassaine diterpenoids. MTT assay confirmed that compound 1, erythrophlesin H, had the strongest cytotoxic effect toward the human prostate cancer cell line, PC-3. The molecular mechanism by which this compound induced apoptosis cell in prostate cancer remains unknown. Erythrophlesin H induced apoptosis in a dose-dependent manner. Acridine orange and annexin V-FITC/PI double staining confirmed that erythrophlesin H effectively induces apoptosis in PC-3 cells.     

Antiproliferative effect of normal and 13-epi-D-homoestrone and their 3-methyl ethers on human reproductive cancer cell lines

The possibility of the therapeutic use of estrogens emerged following the recognition that certain estradiol analogs, and particularly metabolites (e.g. the A-ring metabolite 2-hydroxyestrone, etc.) inhibit the differentiation of diverse tumor cell lines. Until recently, despite the investigation of numerous synthetic d-ring-substituted estrone derivatives, no analysis had been published on the effects of D-ring expansion of estrone on its tumor-suppressing activity. The aim of the present study was to characterize the antiproliferative effects of normal and 13-epi-D-homoestrone and their 3-methyl ethers (1-4) on human reproductive cancer cell lines. The antitumor activities of the two epimer pairs on HeLa, MCF-7 and Ishikawa cells were determined. Normal D-homoestrone exerted the greatest cytostatic effect on HeLa cells (IC(50)=5.5 μM) and was subjected to further investigations to elucidate its mechanism of action on apoptosis induction. Morphological changes detected by Hoechst 33258-propidium iodide double staining, the cell cycle arrest at phase G2/M and the subsequent increase in the proportion of the subG1 fraction determined by flow cytometric analysis and the significant increase in the activity of caspase-3 confirmed the induction of apoptosis in HeLa cells treated with D-homoestrone. D-Homoestrone was also tested on a non-cancerous human lung fibroblast cell line (MRC-5) to determine its selective toxicity. The concentration in which it inhibited cell proliferation by 50% was at least six times higher for the fibroblast cells than for cervical cancer cells. No significant in vivo estrogenic activity was observed as concerns the uterus weight of gonadectomized rats after a 7-day treatment with normal D-homoestrone. These results led to the conclusion that normal D-homoestrone is a novel antitumor compound with a similar activity on HeLa cells as that of the reference agent cisplatin, but its selectivity toward non-cancerous cells is significantly higher than that of cisplatin. It may be considered to be a basic lead molecule for the preclinical development of potential anticancer agents.     

Synthesis of [3β-acetoxy-urs-12-en-28-oyl]-1-monoglyceride and investigation on its anti tumor effects against BGC-823

Ursolic acid (UA) as the leader compound was designed to prepare a series of derivatives (three novel compounds UA-1a, UA-1b and UA-2) by modification at the C3 and C28 positions. Their chemical structures were confirmed by IR, (1)H NMR and MS. The cytotoxic activity of the derivatives was evaluated against HepG2, BGC-823 and HT-29 by the MTT assay. The novel derivative UA-1a, [3β-acetoxy-urs-12-en-28-oyl]-1-monoglyceride showed significant anti-growth ability against the assayed cancer cell lines, particularly against BGC-823, while low cytotoxicity to human normal gastric cell line GES-1. Further investigation revealed that UA-1a could induce apoptotic events of the treated BGC-823 cells, such as comet-like DNA bend, sub-G0/G1 phase accumulation and phosphatidylserine externalization. The activity of Caspase-3 was found to be up-regulated, while the expression of Bcl-2 and Survivin were down-regulated in UA-1a treated cells. UA-1a might trigger the death of BGC-823 cells by inducing apoptosis via the mitochondria pathway. UA-1a exerted stronger ability than Taxol to retard tumor growth in nude mice without leaving apparent toxicity to the hosts. The experimental data suggested that UA-1a would have a therapeutic potential in the treatment of gastric cancer.     

Oxineur,a novel peptide from Caspian cobra Naja naja oxiana against HT-29 colon cancer

Colon cancer ranks fourth in mortality. This cancer is still an important clinical challenge worldwide due to its high prevalence and poor prognosis. Proteomic studies revealed that snake venom is a diverse and variable mixture of enzymatic and non-enzymatic proteins and peptides. Despite the toxic effects of these molecules, several proteins and peptides have been isolated that have practical applications and appear to induce apoptosis and prevent cell metastasis. In this study, we worked on cytotoxic effects and anticancer activity of Naja naja oxiana (Iranian Caspian cobra) snake venom components on HT-29 cell line colon cancer. Separated Fraction-5 by FPLC indicated the high cytotoxicity on HT-29 cell line colon cancer by MTT test. Further isolation of F5 by HPLC showed that the purified peak 2, nominated as Oxineur that contains a cytotoxic effect on HT-29 cells and reduces cell viability at 8 μg/ml to 4% in 24 h. Oxineur has the least cytotoxic effect on HEK-293 normal cells. Further studies on Oxineur peptide confirmed the apoptotic effects with high expression of CASP9 gene and DNA fragmentation in cancerous cells. The partial sequence of Oxineur revealed 71% homology with the neurotoxin II from Naja naja oxiana. Since our target molecule is a peptide in the molecular weight range of 7 kDa, it has potentially a therapeutic value.     

Synthesis and biological evaluation of benzo[b]furo[3,4-e][1,4]diazepin-1-one derivatives as anti-cancer agents

A new series of novel Podophyllotoxin-like benzo[b]furo[3,4-e][1,4]diazepin-1-ones possessing structural elements of 4-aza-2,3-didehydropodophyllotoxins with central diazepine ring was designed and synthesized as anti-cancer agents. In initial assessment, the cytotoxic activity of the synthesized compounds was evaluated against three cancer cell lines including MCF-7, PC3 and B16-F10 employing the MTT assay. Some of compounds (12h, 13a, 13c and 14b) showed significant cytotoxic activity. So, we investigated the cytotoxicity of compounds 12h, 13a, 13c and 14b, along with podophyllotoxin as the reference drug in different cancer cell lines including A549, A2780, DU145, HeLa, and normal Huvec cell line. Among these four compounds, 13c showed promising antiproliferative activity against all cancer cells stronger than the other compounds and comparable to reference drug podophyllotoxin in some cancer cells. All these four compounds did not show significant cytotoxicity on normal Huvec cell line. The flow cytometry analysis of the MCF-7, PC3 and A2780 human cancer cell lines treated with 13c showed that 13c, induced apoptosis in the MCF-7, PC3 and A2780 human cancer cell lines, which is in good agreement to its cytotoxic activity as well. Compound 13c did not show significant influence on tubulin assembly and exert its cytotoxic effects via induction of apoptosis and has potent and selective cytotoxic effects in cancer cells.     

Cytotoxic clerodane diterpenoids from the leaves of Casearia kurzii

A phytochemical investigation to obtain bioactive substances as lead compounds or agents for cancer led to the obtainment of six new clerodane diterpenoids, designated as kurzipenes A–F (1–6), from the leaves of Casearia kurzii. Their structures were elucidated on the basis of NMR spectroscopic data analysis and the absolute configurations were confirmed by the time-dependent density functional theory (TDDFT) electronic circular dichroism (ECD) calculations. The cytotoxic activities of compounds 1–6 were evaluated against human lung cancer A549 cell line, human cervical cancer Hela cell line, and human hepatocellular carcinoma HepG2 cell line. Most diterpenoids showed potent cytotoxicities against the three selected cancer cell lines. The preliminary mechanism studies revealed that the most active compound 2, with an IC₅₀ value of 5.3 μM against Hela cells, induced apoptosis and arrested the Hela cell cycle at the G0/G1 stage to exert cytotoxic effects.     

Anti-proliferative activity of disulfiram through regulation of the AKT-FOXO axis: A proteomic study of molecular targets

Due to its potent anti-tumor activity, well-investigated pharmacokinetic properties and safety profile, disulfiram (DSF) has emerged as a promising candidate for drug repurposing in cancer therapy. Although several molecular mechanisms have been proposed for its anti-cancer effects, the precise underlying mechanisms remain unclear. In the present study, we showed that DSF inhibited proliferation of cancer cells by inducing reactive oxygen species (ROS) production, a G1 cell cycle arrest and autophagy. Moreover, DSF triggered apoptosis via suppression of the anti-apoptotic protein survivin.To elucidate the mechanisms for the anti-proliferative activities of DSF, we applied a 2-DE combined with MALDI-TOF-MS/MS analysis to identify differentially expressed proteins in breast cancer cells upon treatment with DSF. Nine differentially expressed proteins were identified among which, three candidates including calmodulin (CaM), peroxiredoxin 1 (PRDX1) and collagen type I alpha 1 (COL1A1) are involved in the regulation of the AKT signaling pathway. The results of western blot analysis confirmed that DSF inhibited p-AKT, suggesting that DSF induces its anti-tumor effects via AKT blockade. Moreover, we found that DSF increased the mRNA levels of FOXO1, FOXO3 and FOXO4, and upregulated the expression of their target genes involved in G1 cell cycle arrest, apoptosis and autophagy. Finally, DSF potentiated the anti-proliferative effects of well-known chemotherapeutic agents such as arsenic trioxide (ATO), doxorubicin, paclitaxel and cisplatin. Altogether, these findings provide mechanistic insights into the anti-growth activities of DSF.     

Traditional Aboriginal Preparation Alters the Chemical Profile of Carica papaya Leaves and Impacts on Cytotoxicity towards Human Squamous Cell Carcinoma

Carica papaya leaf decoction, an Australian Aboriginal remedy, has been used widely for its healing capabilities against cancer, with numerous anecdotal reports. In this study we investigated its in vitro cytotoxicity on human squamous cell carcinoma cells followed by metabolomic profiling of Carica papaya leaf decoction and leaf juice/brewed leaf juice to determine the effects imparted by the long heating process typical of the Aboriginal remedy preparation. MTT assay results showed that in comparison with the decoction, the leaf juice not only exhibited a stronger cytotoxic effect on SCC25 cancer cells, but also produced a significant cancer-selective effect as shown by tests on non-cancerous human keratinocyte HaCaT cells. Furthermore, evidence from testing brewed leaf juice on these two cell lines suggested that the brewing process markedly reduced the selective effect of Carica papaya leaf on SCC25 cancer cells. To tentatively identify the compounds that contribute to the distinct selective anticancer activity of leaf juice, an untargeted metabolomic approach employing Ultra High Performance Liquid Chromatography-Quadrupole Time of Flight-Mass Spectrometry followed by multivariate data analysis was applied. Some 90 and 104 peaks in positive and negative mode respectively were selected as discriminatory features from the chemical profile of leaf juice and >1500 putative compound IDs were obtained via database searching. Direct comparison of chromatographic and tandem mass spectral data to available reference compounds confirmed one feature as a match with its proposed authentic standard, namely pheophorbide A. However, despite pheophorbide A exhibiting cytotoxic activity on SCC25 cancer cells, it did not prove to be the compound contributing principally to the selective activity of leaf juice. With promising results suggesting stronger and more selective anticancer effects when compared to the Aboriginal remedy, Carica papaya leaf juice warrants further study to explore its activity on other cancer cell lines, as well as investigation to confirm the identity of compounds contributing to its selective effect, particularly those compounds altered by the long heating process applied during the traditional Aboriginal remedy preparation.     

Synthesis,biological evaluation,and in silico studies of new CDK2 inhibitors based on pyrazolo[3,4-d]pyrimidine and pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine scaffold with apoptotic activity

Cyclin-dependent kinase inhibition is considered a promising target for cancer treatment for its crucial role in cell cycle regulation. Pyrazolo pyrimidine derivatives were well established for their antitumor activity via CDK2 inhibition. In this research, new series of pyrazolopyrimidine derivatives (4–15) was designed and synthesised as novel CDK2 inhibitors. The anti-proliferative activities against MCF-7, HCT-116, and HepG-2 were used to evaluate their anticancer activity as novel CDK2 inhibitors. Most of the compounds showed superior cytotoxic activity against MCF-7 and HCT-116 compared to Sorafenib. Only compounds 8, 14, and 15 showed potent activity against HepG-2. The CDK2/cyclin A2 enzyme inhibitory activity was tested for all synthesised compounds. Compound 15 showed the most significant inhibitory activity with IC₅₀ 0.061 ± 0.003 µM. It exerted remarkable alteration in Pre G1 and S phase cell cycle progression and caused apoptosis in HCT cells. In addition, the normal cell line cytotoxicity for compound 15 was assigned revealing low cytotoxic results in normal cells rather than cancer cells. Molecular docking was achieved on the designed compounds and confirmed the two essential hydrogen binding with Leu83 in CDK2 active site. In silico ADMET studies and drug-likeness showed proper pharmacokinetic properties which helped in structure requirements prediction for the observed antitumor activity.     

A novel adamantane thiadiazole derivative induces mitochondria-mediated apoptosis in lung carcinoma cell line

The interaction of organic compounds with apoptosis regulatory proteins is an attractive field of research because of its relevance in the development of new chemotherapeutic agents for cancer treatment. Our group designed four new adamantane thiadiazole derivatives (ATDs). The four ATDs were theoretically tested for their binding affinities to a model of an apoptosis inhibitor protein using molecular modeling. ATD-4 which interacted with the highest binding affinity was synthesized and characterized. The in vitro cytotoxicity of ATD-4 against different cancer cell lines as well as normal cell line was determined and compared with 5-fluorouracil as a standard positive control. The lung carcinoma cell line that showed the highest cytotoxic activity due to ATD-4 treatment was chosen to further study if ATD-4 can perform its cytotoxic activity through the induction of apoptosis as expected from molecular modeling. Inducing apoptosis by ATD-4 in lung carcinoma cell line was assessed by various biochemical and morphological characteristics. Biochemically: The effect of ATD-4 on cell cycle and its ability to induce apoptosis were checked through flow cytometry. Caspase-3 activity was detected by a colorimetric method. Real time-polymerase chain reaction (q-PCR) was used to detect p53, caspase-3, bcl-2 and bax gene expression. Morphologically: Changes in cell surface morphology, granulation and average surface roughness were detected using atomic force microscopy (AFM). Cell shrinkage, increase in cytoplasmic organelles, changes in mitochondrial number and morphology, chromatin condensation, membrane blebbing and formation of apoptotic bodies were detected using transmission electron microscopy (TEM). The obtained results suggest that ATD-4 exerted its antitumor activity against A549 cells through the induction of the intrinsic (mitochondrial) apoptotic pathway.     

Effects of karanjin on cell cycle arrest and apoptosis in human A549, HepG2 and HL-60 cancer cells

Background

We have investigated the potential anticancer effects of karanjin, a principal furanoflavonol constituent of the Chinese medicine Fordia cauliflora, using cytotoxic assay, cell cycle arrest, and induction of apoptosis in three human cancer cell lines (A549, HepG2 and HL-60 cells).

Results

MTT cytotoxic assay showed that karanjin could inhibit the proliferation and viability of all three cancer cells. The induction of cell cycle arrest was observed via a PI (propidium iodide)/RNase Staining Buffer detection kit and analyzed by flow cytometry: karanjin could dose-dependently induce cell cycle arrest at G2/M phase in the three cell lines. Cell apoptosis was assessed by Annexin V-FITC/PI staining: all three cancer cells treated with karanjin exhibited significantly increased apoptotic rates, especially in the percentage of late apoptosis cells.

Conclusion

Karanjin can induce cancer cell death through cell cycle arrest and enhance apoptosis. This compound may be effective clinically for cancer pharmacotherapy.     

Synthesis of substituted biphenyl methylene indolinones as apoptosis inducers and tubulin polymerization inhibitors

A new series of biphenyl methylene indolinones has been designed, synthesized and evaluated for their in vitro antiproliferative activity against various cancer cell lines like DU-145 (prostate cancer cell line), 4T1 (mouse breast cancer cell line), MDA-MB-231 (human breast cancer cell line), BT-549 (human breast cancer cell line), T24 (human urinary bladder carcinoma cell line), and HeLa (cervical cancer cell line). Among the series, compound 10e showed potent in vitro cytotoxic activity against HeLa and DU-145 cancer cell lines with IC₅₀ value of 1.74 ± 0.69 µM and 1.68 ± 1.06 µM respectively. To understand the underlying mechanism of most potent cytotoxic compound 10e, various mechanistic studies were carried out on DU-145 cell lines. Cell cycle analysis results revealed that these conjugates affect both G0/G1 and G2/M phase of the cycle, tubulin binding assay resulted that compound 10e interrupting microtubule network formation by inhibiting tubulin polymerization with IC₅₀ value of 4.96 ± 0.05 μM. Moreover, molecular docking of 10e on colchicine binding site of the tubulin explains the interaction of 10e with tubulin. Clonogenic assay indicated inhibition of colony formation by compound 10e in a dose dependent manner. In addition, morphological changes were clearly observed by AO/EB and DAPI staining studies. Moreover, ROS detection using DCFDA, JC-1, and annexin V-FITC assays demonstrated the significant apoptosis induction by 10e.     

The cytotoxic effect of 2-acylated-1,4-naphthohydroquinones on leukemia/lymphoma cells

Here, we tested seven 2-acylated-1,4-hydronaphthoquinones for their cytotoxic effects on a panel of cancer lymphoma/leukemia cells and compared to a non-cancer origin cell line. Several naphthohydroquinones exhibited selective cytotoxic effects on lymphoma/leukemia cells with lowest activity on non-cancer cells. The mode of cell death induced by an acylated naphthohydroquinone, which has a long alkyl chain, was found to be via apoptosis. Furthermore, the naphthohydroquinone provoked mitochondria depolarization and activation of its downstream effector, caspase-3, thus implicating the intrinsic apoptotic pathway as its mechanism to exert cell death.     

Immunomodulation-mediated anticancer activity of a novel compound from Brugmansia suaveolens leaves

Immunomodulation activity-guided fractionation of ethanol extract of Brugmansia suaveolens leaves was carried out to isolate a novel compound SUPH036-022A (1) by co-culturing the test fraction/compound activated PBMC with MCF7 and A549 cancer cell lines. Assessment of immune markers in PBMC, and analysis of apoptosis markers and cell cycle was carried out for cancer cells. The structure of the isolated compound was elucidated by spectral analysis. Compound 1 enhanced the secretion of immune markers, IL-2 and IFN-γ, from PBMC. Further, compound 1 treated PBMC increased cell death in MCF7 and A549 cell lines and induced ROS production and mitochondrial membrane perturbation, leading to apoptosis. Flow cytometry analysis revealed; compound 1 stimulated PBMC to cause a five-fold increase in cell cycle perturbations in the sub-G1 stage of cancer cells as compared to the negative control. The compound, in the absence of PBMC, only had a weak cytotoxic activity against these cell lines. Thus, compound 1 is a novel lead for immunomodulation-mediated anticancer activity.     

Cytotoxicity and apoptosis induction of Bacillus vallismortis BIT-33 metabolites on colon cancer carcinoma cells

Aims:  The objective of this research was to isolate and identify a cytotoxic marine bacterium, BIT-33, and to investigate the apoptosis effects of its metabolite on colon cancer cells.
Method and Results:  We isolated 93 marine bacteria from seawater samples. Of these, strain BIT-33 exhibited the strongest cytotoxic activity on three colon cancer cells (HT-29, SW480 and HCT116). Biochemical tests and 16S rDNA sequencing of this strain allowed us to identify BIT-33 as a strain of Bacillus vallismortis . The cytotoxic compound from B. vallismortis BIT-33 was purified by reverse-phase high-performance liquid chromatography. Direct cytotoxic effect of the compound was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide assay. The compound induced apoptosis of colon cancer cells, as indicated by DNA fragmentation of agarose gel electrophoresis, flow cytometric analysis (sub-G₁ method) and annexin V staining.
Conclusion:  The cytotoxic compound from B. vallismortis BIT-33 was purified, and the compound showed direct cytotoxic and apoptotic effects on colon cancer cells in a dose- and time-dependent manner.
Significance and Impact of the Study:  Taken together, our results suggest that the compound from B. vallismortis BIT-33 could be a candidate for the development of apoptosis-specific anti-tumour agents. This study indicated that marine bacteria could be an important source of cytotoxic metabolites.     

Cytotoxic and apoptotic activities of Amorphophallus campanulatus (Roxb.) Bl. tuber extracts against human colon carcinoma cell line HCT-15

Colorectal cancer is one of the leading causes of cancer death worldwide and is the third most common form of malignancy in both men and women. Several possible colon cancer chemopreventive agents are found in edible plants. Amorphophallus campanulatus (Roxb.) Blume (family: Araceae) is a tuber crop, largely cultivated throughout the plains of India for using its corm as food. This tuber has also been traditionally used for the treatment of abdominal tumors, liver diseases, piles etc. The aim of this study was to evaluate the dose-dependent cytotoxic and apoptosis inducing effects of the sub fractions of A. campanulatus tuber methanolic extract (ACME) viz. petroleum ether fraction (PEF), chloroform fraction (CHF), ethyl acetate fraction (EAF) and methanolic fraction (MEF) on the colon cancer cell line, HCT-15. Antiproliferative effects of the sub fractions of ACME were studied by MTT assay. Apoptotic activity was assessed by DAPI, Annexin V-FITC and JC-1 fluorescent staining. The chemotherapeutic drug, 5-flurouracil (5-FU) was used as positive drug control. The sub fractions of ACME significantly inhibited the proliferation of HCT-15 cells in a dose-dependent manner. In addition, the extracts were found to induce apoptosis and were confirmed by DAPI, Annexin V-FITC and JC-1 fluorescent staining. A pronounced results of cytotoxic and apoptotic activities were observed in the cells treated with 5-FU and CHF, whereas, EAF and MEF treated cells exhibited a moderate result and the least effect was observed in PEF treated cells. Our results suggested that, among the sub fractions of ACME, CHF had potent cytotoxic and apoptotic activity and thus it could be explored as a novel target for anticancer drug development. Furthermore, these findings confirm that the sub fractions of ACME dose-dependently suppress the proliferation of HCT-15 cells by inducing apoptosis.     

Synthesis and in vitro antitumor activity of novel alkenyl derivatives of pyridoxine,bioisosteric analogs of feruloyl methane

Two series of novel pyridoxine-based azaheterocyclic analogs of feruloyl methane (Dehydrozingerone, DZG) were synthesized, and their biological activity against a panel of tumor and normal cell lines was evaluated in vitro. The most active compounds possessed expressed cytotoxic activity, which was comparable to cytotoxic activity of doxorubicin and significantly higher than that of DZG, and a remarkable selectivity for the studied cancer cell lines as compared to the normal cells. The leading compound and DZG initiated arrest of the cell cycle in the G2/M phase, preventing normal division and further transition of daughter cells to the G0/G1 phase. Similar to DZG, but with higher efficiency, the leading compound was able to inhibit migration activity and, therefore, invasiveness of tumor cells. It also increased concentration of reactive oxygen species in tumor cells, induced depolarization of mitochondrial membranes and initiated apoptosis accompanied by disruption of integrity of cytoplasmic cell membranes. By contrast to DZG, the leading compound did not possess antioxidant properties. The obtained data make the described chemotype a promising starting point for the development of new anticancer agents.     

A trans-platinum(II) complex induces apoptosis in cancer stem cells of breast cancer

Recent accumulating evidence has supported the notion that tumors have hierarchically organized heterogeneous cell populations and a small subpopulation of cells, termed cancer stem cells (CSCs), are responsible for tumor initiation, maintenance as well as drug resistance. Therefore, targeting the CSCs along with the other cancer cells has been the most important topic during the last decade. In the present study, we evaluated the cytotoxic activity of trans-[PtCl₂(2-hepy)₂] [2-hepy = 2-(2-hydroxyethyl) pyridine] complex and the mechanism of cell death in breast CSCs. Stemness markers, Oct-4 and Sox2, were determined in mammospheres by western blotting. Cytotoxicity was assessed using the ATP viability assay. Cell death was fluorescently visualized and further confirmed by flow cytometry as well as gene expression analysis. The Pt(II) complex significantly reduced the cell viability, prevented mammosphere formation and disrupted mammosphere structures in a dose-dependent manner (0–100 μM). The mode of cell death was apoptosis and it was shown by the presence of caspase 3/7 activity, Annexin V-FITC positivity, decreased mitochondrial membrane potential and increased expressions of pro-apoptotic genes (TNFRSF10A and HRK). Interestingly, necroptosis was also observed by the evidence of increased MLKL expression. In conclusion, the Pt(II) complex seems to be a highly promising anticancer compound due to its promising cytotoxic activity on CSCs. Therefore, it deserves in vivo further studies for the proof-of-concept.