Receptor-interacting protein kinase 2 (RIPK2) stabilizes c-Myc and is a therapeutic target in prostate cancer metastasis.

Despite progress in prostate cancer (PC) therapeutics, distant metastasis remains a major cause of morbidity and mortality from PC. Thus, there is growing recognition that preventing or delaying PC metastasis holds great potential for substantially improving patient outcomes. Here we show receptor-interacting protein kinase 2 (RIPK2) is a clinically actionable target for inhibiting PC metastasis. RIPK2 is amplified/gained in ~65% of lethal metastatic castration-resistant PC. Its overexpression is associated with disease progression and poor prognosis, and its genetic knockout substantially reduces PC metastasis. Multi-level proteomics analyses reveal that RIPK2 strongly regulates the stability and activity of c-Myc (a driver of metastasis), largely via binding to and activating mitogen-activated protein kinase kinase 7 (MKK7), which we identify as a direct c-Myc-S62 kinase. RIPK2 inhibition by preclinical and clinical drugs inactivates the noncanonical RIPK2/MKK7/c-Myc pathway and effectively impairs PC metastatic outgrowth. These results support targeting RIPK2 signaling to extend metastasis-free and overall survival.

Reciprocal deregulation of NKX3.1 and AURKA axis in castration-resistant prostate cancer and NEPC models.

BACKGROUND: NKX3.1, a prostate-specific tumor suppressor, is either genomically lost or its protein levels are severely downregulated, which are invariably associated with poor prognosis in prostate cancer (PCa). Nevertheless, a clear disconnect exists between its mRNA and protein levels, indicating that its post-translational regulation may be critical in maintaining its protein levels. Similarly, AURKA is vastly overexpressed in all stages of prostate cancer (PCa), including castration-resistant PCa (CRPC) and neuroendocrine PCa (NEPC), although its transcripts are only increased in ~ 15% of cases, hinting at additional mechanisms of deregulation. Thus, identifying the upstream regulators that control AURKA and NKX3.1's levels and/or their downstream effectors offer an alternative route to inhibit AURKA and upregulate NKX3.1 in highly fatal CRPC and NEPC. AURKA and NKX3.1 have not linked to each other in any study to date. METHODS: A chemical genetic screen revealed NKX3.1 as a direct target of AURKA. AURKA-NKX3.1 cross-talk was analyzed using several biochemical techniques in CRPC and NEPC cells. RESULTS: We uncovered a reciprocal loop between AURKA and NKX3.1 in CRPC and NEPC cells. We observed that AURKA-mediated NKX3.1 downregulation is a major mechanism that drives CRPC pathogenesis and NEPC differentiation. AURKA phosphorylates NKX3.1 at three sites, which degrades it, but AURKA does not regulate NKX3.1 mRNA levels. NKX3.1 degradation drives highly aggressive oncogenic phenotypes in cells. NKX3.1 also degrades AURKA in a feedback loop. NKX3.1-AURKA loop thus upregulates AKT, ARv7 and Androgen Receptor (AR)-signaling in tandem promoting highly malignant phenotypes. Just as importantly, we observed that NKX3.1 overexpression fully abolished synaptophysin and enolase expression in NEPC cells, uncovering a strong negative relationship between NKX3.1 and neuroendocrine phenotypes, which was further confirmed be measuring neurite outgrowth. While WT-NKX3.1 inhibited neuronal differentiation, 3A-NKX3.1 expression obliterated it. CONCLUSIONS: NKX3.1 loss could be a major mechanism causing AURKA upregulation in CRPC and NEPC and vice versa. NKX3.1 genomic loss requires gene therapy, nonetheless, targeting AURKA provides a powerful tool to maintain NKX3.1 levels. Conversely, when NKX3.1 upregulation strategy using small molecules comes to fruition, AURKA inhibition should work synergistically due to the reciprocal loop in these highly aggressive incurable diseases.

Novel α-tocopherol-ferrocene conjugates for the specific delivery of transgenes in liver cancer cells under high serum conditions.

The delivery of therapeutic genes to a specific organ has drawn significant research attention. Among the pool of various delivery vectors, cationic liposomes (non-viral) are potential candidates for delivering therapeutic genes due to their low immunogenic response. Here, we have developed novel ferrocene-conjugated cationic tocopheryl aggregates as non-viral vectors. These formulations can transfer a reporter gene (pGL3; encoded for luciferase protein) specifically to liver cancer cells (HepG2 and Huh7) instead of non-hepatic cancer cells, such as Caco-2 (human colon carcinoma) and HeLa (cervical cancer) cells. The transfection efficiency (TE) of the optimum liposomal formulation is more significant than commercially available Lipofectamine 2000 (L2K). Notably, it retains its TE under high serum conditions (up to 50% FBS). A coupled effect from conjugated ferrocene and tocopherol in the cationic liposomal formulation might be responsible for the cell-specific delivery and higher serum compatibility. Therefore, the present proposed delivery system may provide a platform for further progress in terms of developing hepatotropic gene delivery systems.

LIMK2-NKX3.1 Engagement Promotes Castration-Resistant Prostate Cancer.

NKX3.1's downregulation is strongly associated with prostate cancer (PCa) initiation, progression, and CRPC development. Nevertheless, a clear disagreement exists between NKX3.1 protein and mRNA levels in PCa tissues, indicating that its regulation at a post-translational level plays a vital role. This study identified a strong negative relationship between NKX3.1 and LIMK2, which is critical in CRPC pathogenesis. We identified that NKX3.1 degradation by direct phosphorylation by LIMK2 is crucial for promoting oncogenicity in CRPC cells and in vivo. LIMK2 also downregulates NKX3.1 mRNA levels. In return, NKX3.1 promotes LIMK2's ubiquitylation. Thus, the negative crosstalk between LIMK2-NKX3.1 regulates AR, ARv7, and AKT signaling, promoting aggressive phenotypes. We also provide a new link between NKX3.1 and PTEN, both of which are downregulated by LIMK2. PTEN loss is strongly linked with NKX3.1 downregulation. As NKX3.1 is a prostate-specific tumor suppressor, preserving its levels by LIMK2 inhibition provides a tremendous opportunity for developing targeted therapy in CRPC. Further, as NKX3.1 downregulates AR transcription and inhibits AKT signaling, restoring its levels by inhibiting LIMK2 is expected to be especially beneficial by co-targeting two driver pathways in tandem, a highly desirable requisite for developing effective PCa therapeutics.

Negative cross talk between LIMK2 and PTEN promotes castration resistant prostate cancer pathogenesis in cells and in vivo.

Androgen deprivation therapy (ADT) and androgen receptor (AR) signaling inhibitors are front-line treatments for highly aggressive prostate cancer. However, prolonged inhibition of AR triggers a compensatory activation of PI3K pathway, most often due to the genomic loss of tumor suppressor PTEN, driving progression to the castration-resistant prostate cancer (CRPC) stage, which has very poor prognosis. We uncovered a novel mechanism of PTEN downregulation triggered by LIMK2, which contributes significantly to CRPC pathogenesis. LIMK2 is a CRPC-specific target. Its depletion fully reverses tumorigenesis in vivo. LIMK2 phosphorylates PTEN at five sites, degrading and inhibiting its activity, thereby driving highly aggressive oncogenic phenotypes in cells and in vivo. PTEN also degrades LIMK2 in a feedback loop, which was confirmed in prostates from PTEN-/- and PTEN+/+ mice. LIMK2 is also the missing link between hypoxia and PTEN degradation in CRPC. This is the first study to show a feedback loop between PTEN and its regulator. Uncovering the LIMK2-PTEN loop provides a powerful therapeutic opportunity to retain the activity and stability of PTEN protein by inhibiting LIMK2, thereby halting the progression to CRPC, ADT-resistance and drug-resistance.

Phosphorylation-dependent regulation of SPOP by LIMK2 promotes castration-resistant prostate cancer.

BACKGROUND: SPOP, an E3 ubiquitin ligase adaptor, can act either as a tumour suppressor or a tumour promoter. In prostate cancer (PCa), it inhibits tumorigenesis by degrading several oncogenic substrates. SPOP is the most altered gene in PCa (~15%), which renders it ineffective, promoting cancer. The remaining PCa tumours, which retain WT-SPOP, still progress to castration-resistant (CRPC) stage, indicating that other critical mechanisms exist for downregulating SPOP. SPOP is reduced in ~94% of WT-SPOP-bearing prostate tumours; however, no molecular mechanism is known for its downregulation. METHODS: SPOP was identified as a direct target of LIMK2 using an innovative technique. The reciprocal relationship between SPOP and LIMK2 and its consequences on oncogenicity were analysed using a variety of biochemical assays. To probe this relationship in vivo, xenograft studies were conducted. RESULTS: LIMK2 degrades SPOP by direct phosphorylation at three sites. SPOP promotes LIMK2's ubiquitylation, creating a feedback loop. SPOP's degradation stabilises AR, ARv7 and c-Myc promoting oncogenicity. Phospho-resistant SPOP completely suppresses tumorigenesis in vivo, indicating that LIMK2-mediated SPOP degradation is a key event in PCa progression. CONCLUSIONS: While genomically altered SPOP-bearing tumours require gene therapy, uncovering LIMK2-SPOP relationship provides a powerful opportunity to retain WT-SPOP by inhibiting LIMK2, thereby halting disease progression.

Molecular Interplay between AURKA and SPOP Dictates CRPC Pathogenesis via Androgen Receptor.

SPOP, an adaptor protein for E3 ubiquitin ligase can function as a tumor-suppressor or a tumor-enhancer. In castration-resistant prostate cancer (CRPC), it inhibits tumorigenesis by degrading many oncogenic targets, including androgen receptor (AR). Expectedly, SPOP is the most commonly mutated gene in CRPC (15%), which closely correlates with poor prognosis. Importantly, 85% of tumors that retain wild-type SPOP show reduced protein levels, indicating that SPOP downregulation is an essential step in CRPC progression. However, the underlying molecular mechanism remains unknown. This study uncovered the first mechanism of SPOP regulation in any type of cancer. We identified SPOP as a direct substrate of Aurora A (AURKA) using an innovative technique. AURKA directly phosphorylates SPOP at three sites, causing its ubiquitylation. SPOP degradation drives highly aggressive oncogenic phenotypes in cells and in vivo including stabilizing AR, ARv7 and c-Myc. Further, SPOP degrades AURKA via a feedback loop. SPOP upregulation is one of the mechanisms by which enzalutamide exerts its efficacy. Consequently, phospho-resistant SPOP fully abrogates tumorigenesis and EMT in vivo, and renders CRPC cells sensitive to enzalutamide. While genomic mutations of SPOP can be treated with gene therapy, identification of AURKA as an upstream regulator of SPOP provides a powerful opportunity for retaining WT-SPOP in a vast majority of CRPC patients using AURKA inhibitors ± enzalutamide, thereby treating the disease and inhibiting its progression.

Specific stabilization of promoter G-Quadruplex DNA by 2,6-disubstituted amidoanthracene-9,10-dione based dimeric distamycin analogues and their selective cancer cell cytotoxicity.

We have designed and synthesized anthraquinone containing compounds which have oligopyrrole side chains of varying lengths. These compounds stabilized the G-quadruplex DNA formed in the promoter regions of c-MYC oncogenes selectively over the duplex DNA. These observations were recorded using UV-vis spectroscopic titrations, fluorescence measurements and circular dichroism (CD) spectral titrations. The potency of the compounds to stabilize the G4 DNA has been shown from the thermal denaturation experiments. The compound interacts with c-MYC G-quadruplex DNA through stacking mode as obtained from ethidium bromide displacement assay, cyclic voltammetric titration, and docking experiments. Molecular modeling studies suggested that the stacking of the anthraquinone moiety over the G-tetrad of the G4 structures are responsible for the stability of such quadruplex secondary structure. Furthermore, polymerase stop assay also supported the formation of stable G4 structures in the presence of the above-mentioned compounds. The compounds have shown selective cancer cell (HeLa and HEK293T) cytotoxicity over normal cells (NIH3T3 and HDFa) under in vitro conditions as determined from MTT based cell viability assay. Apoptosis was found to be the mechanistic pathway underlying the cancer cell cytotoxicity as obtained from Annexin V-FITC and PI dual staining assay which was further substantiated by nuclear morphological changes as observed by AO/EB dual staining assay. Cellular morphological changes, as well as nuclear condensation and fragmentation upon treatment with these compounds, were observed under bright field and confocal microscopy.

Antibody-Conjugated Vitamin E-Derived Liposomes for Targeted Gene Transfer.

Construction of a vitamin E-based liposomal biomaterial and its ability to deliver therapeutic genes selectively across liver cancer cells are demonstrated herein. In humans, liver regulates the levels of α-tocopherol, i.e., vitamin E, and hepatic cells carry the machinery for its transport. To exploit the presence of tocopherol transport protein, we have selected an efficient gene transfecting α-tocopherol-based twin lipid bearing a hydroxyethylated headgroup and octamethylene spacer (TH8S) for liposome formation. Also, based on the abundancy of the low-density lipoprotein receptor (LDLr) on the cellular surface in the case of hepatocellular carcinoma, anti-LDLr monoclonal antibody is used to confer the targeting ability to liposomes. A facile thiol-maleimide click chemistry is used for antibody decoration on the liposomal surface. Selective delivery of reporter and therapeutic genes (GFP and p53) to cells of hepatic origin was observed using anti-LDLr-tagged TH8S liposomes. Cellular internalization by receptor-mediated endocytosis renders the bioconjugate highly specific as well as highly efficient. Compatibility of the designed material with human blood points to its safety of use in systemic circulation thereby highlighting its in vivo potential. Thus, we report here a versatile biomaterial derived from an essential vitamin that promises potential for targeted suicidal gene therapy.

New Water-Soluble Oxyamino Chitosans as Biocompatible Vectors for Efficacious Anticancer Therapy via Co-Delivery of Gene and Drug.

Among the many nonviral gene delivery vectors, chitosan, being a polysaccharide of natural origin, has gained special importance. In this report, chitosan (CS) has been solubilized in water by preparing its O-carboxymethyl derivative, CS(CH2COOH), with an optimum degree of carboxymethylation. This has been further derivatized to get the pyridine-substituted product (py)CS(CH2COOH), where the degree of pyridine substitution (47%) was optimized based on zeta potential measurements. The optimized formulation showed a high gene binding ability, forming nanosized positively charged polyelectrolyte complexes with DNA. These polyplexes were stable to DNase and physiological polyanions such as heparin. They also exhibited minimal toxicity in vitro and showed transfection levels comparable to the commercial standard Lipofectamine 2000 and much higher than polyethylenimine (MW, 25 kDa). Additionally, in this study, a hitherto unknown oxyamine derivative of chitosan has been prepared by phthaloyl protection, tosylation, and Gabriel's phthalimide synthesis. Nearly 40% of the primary alcohols were successfully converted to oxyamino functionality, which was used for forming oxime with the anticancer drug doxorubicin. The pH sensitivity of the oxime ether linkage and stability under biologically relevant conditions were then used to establish the compound as a versatile drug delivery vector. Co-delivery of functional gene (p53) and drug (doxorubicin) was accomplished in vitro and in vivo with the chitosan-pyridine imine vector (py)CS(CH2COOH) and the newly synthesized doxorubicin oxime ether CS(Dox). Complete tumor regression with no tumor recurrence and appreciable survivability point to the in vivo effectiveness and biocompatibility of the designed composite formulation. Overall, the pH sensitivity of the oxime linkage aiding slow and steady drug release, together with the sustained gene expression by pyridine-tethered carboxymethyl chitosan, allows us to generate a nanobiocomposite with significantly high anticancer therapeutic potential.

Tumor Chemosensitization through Oncogene Knockdown Mediated by Unique α-Tocopherylated Cationic Geminis.

Herein, siRNA transfection efficiency of a unique set of α-tocopherylated gemini lipids has been established in vitro and in vivo. High efficacy of oncogene silencing achieved using the biomacromolecular assembly, formed from siRNA complexes of co-liposomes containing an α-tocopherylated gemini lipid, has been utilized for tumor regression via chemosensitization. Delivery studies with the gemini bearing hydroxyethyl headgroup with octamethylene spacer (TH8S) pointed to a higher siRNA transfection efficacy than its analog without hydroxyethyl group (T8S). Owing to p53 upregulation, transfected cells showed enhanced sensitivity to the chemotherapeutic agent, doxorubicin. Studies in murine model revealed significantly low levels of survivin mRNA in xenograft tumors injected with siRNA lipoplexes, leading to effective inhibition of tumor growth and an increase in sensitivity of the tumors toward doxorubicin. These findings enable us to propose the anti-survivin siRNA carrying TH8S co-liposomes as a potent member of cancer management strategies using suicide gene therapy.

Correction: Transfection efficiencies of α-tocopherylated cationic gemini lipids with hydroxyethyl bearing headgroups under high serum conditions.

Correction for 'Transfection efficiencies of α-tocopherylated cationic gemini lipids with hydroxyethyl bearing headgroups under high serum conditions' by Bappa Maiti et al., Org. Biomol. Chem., 2018, 16, 1983-1993.

Simultaneous sensing of ferritin and apoferritin proteins using an iron-responsive dye and evaluation of physiological parameters associated with serum iron estimation.

An iron-responsive optical probe has been developed for simultaneous sensing of both ferritin and apoferritin proteins at pH 7.4 in water. The compound showed an exclusive response (turn-off signal) towards ferritin among a wide range of proteins even at nanomolar concentration. In contrast, apoferritin dissociates the preformed iron complex and revives the green colored fluorescence of the native probe (turn-on signal). Subsequently, various parameters associated with the serum iron level are evaluated, which are beneficial for clinical diagnosis of many iron-related diseases, including anemia. Estimation of iron was achieved in a wide range of edible plant materials as well as pharmaceutical formulations. Subsequently, different kinds of natural water samples were screened for quantification of soluble iron contents. In addition to traditional spectroscopic tools, dye-coated paper strips were developed as an alternative strategy for onsite 'instrument-free' detection of iron. Highly specific bioimaging of Fe3+ was achieved in cervical cancer cells (HeLa).

Transfection efficiencies of α-tocopherylated cationic gemini lipids with hydroxyethyl bearing headgroups under high serum conditions.

Herein, five new α-tocopheryl cationic gemini lipids with hydroxyethyl bearing headgroups (THnS, n = 4, 5, 6, 8, 12) have been synthesized for efficient plasmid DNA (pDNA) delivery into cancer cells. Among these gemini lipid formulations, the lipid with an octamethylene [-(CH2)8] spacer (TH8S) showed the highest transfection efficiency (TE) that was comparable to that of the commercial standard lipofectamine 2000 (L2K) in terms of luciferase expression in HepG2 (liver hepatocellular carcinoma) cells. The addition of the helper lipid DOPE (1,2-dioleoyl phosphatidyl ethanolamine) with cationic lipids in mixed liposomes further enhanced the TE and the optimized molar ratio was 2 : 1 (DOPE : cationic lipid). The optimized co-liposomal formulation of TH8S (DOPE : TH8S = 2 : 1) showed a higher TE in HepG2, A549 (human lung carcinoma) and MCF7 (human breast adenocarcinoma) cells than other optimized co-liposomal formulations and was also significantly more potent than L2K. The comparison of the TE of DOPE-TH8S (2 : 1) with the gemini lipid T8T (the headgroup devoid of the hydroxyl group) further demonstrated the importance of the hydroxyethyl functionality at the level of the headgroup. Relatively good binding efficiency and easy release of pDNA (pGL3) were also observed with DOPE-TH8S (2 : 1) in the ethidium bromide (EB)-exclusion and re-intercalation assay, which may be the plausible reason for high TE. The lipoplexes were also characterized by atomic force microscopy (AFM), dynamic light scattering (DLS), zeta potential and small angle X-ray diffraction experiments. Greater cellular internalization of fluorescein tagged pDNA was also observed with DOPE-TH8S (2 : 1) lipoplexes compared to that with L2K. Retention of the TE of DOPE-TH8S (2 : 1) lipoplexes under high serum conditions was conferred by the presence of the tocopherol backbone and also the hydroxyethyl functionalities. The cellular internalization pathway of the lipoplexes was characterized by performing transfection experiment in the presence of inhibitors of different endocytic pathways and it was found to be caveolae mediated. An MTT based cell viability assay indicated that the lipoplex mediated gene delivery vectors exhibited low toxicity in all the three cancer cell lines studied.

Identification of a flavonoid isolated from plum (Prunus domestica) as a potent inhibitor of Hepatitis C virus entry.

Hepatitis C virus (HCV) infection is a major cause of chronic liver diseases that often requires liver transplantation. The standard therapies are limited by severe side effects, resistance development, high expense and in a substantial proportion of cases, fail to clear the infection which bespeak the need for development of well-tolerated antivirals. Since most of the drug development strategies target the replication stage of viral lifecycle, the identification of entry inhibitors might be crucial especially in case of liver-transplant recipients. In the present study we have evaluated fruits which are known for their hepatoprotective effects in order to screen for entry inhibitors. We report the identification of a flavonoid, rutin, isolated from Prunus domestica as a new HCV entry inhibitor. Characterization and confirmation of the chemical structure was done by LC-ESI-MS, NMR and IR spectral analyses. Rutin significantly inhibited HCV-LP binding to hepatoma cells and inhibited cell-culture derived HCV (HCVcc) entry into hepatoma cells. Importantly, rutin was found to be non-toxic to hepatoma cells. Furthermore, rutin inhibits the early entry stage of HCV lifecycle possibly by directly acting on the viral particle. In conclusion, rutin is a promising candidate for development of anti-HCV therapeutics in the management of HCV infection.

A plant-derived dehydrorotenoid: a new inhibitor of hepatitis C virus entry.

Emergence of drug-resistant viruses, high cost and adverse side-effects associated with the standard therapy against hepatitis C virus (HCV) infection demonstrate the need for development of well tolerated and effective antivirals. We identified and chemically characterised the dehydrorotenoid boeravinone H, isolated from the herb Boerhavia diffusa, as a new inhibitor of HCV entry. The compound significantly inhibits the binding and entry of hepatitis C-like particles (HCV-LPs) in hepatoma cells in vitro with no apparent cytotoxicity. Boeravinone H inhibits the initial phase of HCV entry probably by acting directly on the viral particle. Importantly, the compound prevents HCV entry and infection in cell culture (ex vivo). Thus, boeravinone H is a potential antiviral agent for the prevention and control of HCV infection.

Enhanced G-Quadruplex DNA Stabilization and Telomerase Inhibition by Novel Fluorescein Derived Salen and Salphen Based Ni(II) and Pd(II) Complexes.

Metal based salen complexes have been considered as an important scaffold toward targeting of DNA structures. In the present work, we have synthesized nickel(II) and palladium(II) salen and salphen complexes by using readily available fluorescein as the backbone to provide an extended aromatic surface. The metal complexes exhibit affinity toward the human telomeric G-quadruplex DNA with promising inhibition of telomerase activity. This has been ascertained by their efficiency in the long term cell proliferation assay which showed significant cancer cell toxicity in the presence of the metal complexes. Confocal microscopy showed cellular internalization followed by localization in the nucleus and mitochondria. Considerable population at the sub-G1 phase of the cell cycle showed cell death via apoptotic pathway.

Novel Oligopyrrole Carboxamide based Nickel(II) and Palladium(II) Salens, Their Targeting of Human G-Quadruplex DNA, and Selective Cancer Cell Toxicity.

DNA targeting by various metal complexes is a key strategy toward the restriction of cancer cell proliferation. Toward this end, we designed and synthesized novel salen-based Ni(II) and Pd(II) metal complexes with positively charged flanking side chains comprising N-methylpyrrole carboxamides of varying lengths. The compounds showed high specificity toward G-quadruplex DNA over duplex DNA. Sufficient inhibition of the telomerase activity was observed, which was ascertained by the prominent restriction of cancer cell proliferation in the long-term cell viability and telomerase inhibition assays. The compounds exhibited selective cancer cell death following an apoptotic pathway. Analysis of the binding mode showed partial stacking of the salen moiety over the G-tetrads and association of the pendant oligopyrrole carboxamide units with the grooves. The conjugation of the tetrad-binding metal salen core with groove-oriented flexible oligopyrrole moieties resulted in the high selectivity and stabilization of the human G-quadruplex DNA structures.

New Fe(iii) and Co(ii) salen complexes with pendant distamycins: selective targeting of cancer cells by DNA damage and mitochondrial pathways.

Minor groove binding distamycin like moieties were conjugated with core salens and the corresponding Fe(iii) and Co(ii) complexes were synthesized. Herein, we have shown efficient DNA minor groove binding specificities along with excellent DNA cleavage capacities with metallosalen conjugates. The metal complexes showed toxicity toward various cancer cells over normal cells with high specificity. Interestingly, the Co(ii) complexes exhibited greater activity than the Fe(iii) complexes in accordance with the stronger affinity of the former in the biophysical studies. Active DNA damage, and prominent nuclear condensation along with the release of cytochrome-c from the mitochondria unanimously showed that the metal complexes followed apoptotic pathways to induce cell death.