Tumoricidal properties of thymoquinone on human colorectal adenocarcinoma cells via the modulation of autophagy.

Colorectal cancer (CRC) is deadly anaplastic changes in the gastrointestinal tract with high-rate mortality. In recent years, the application of phytocompounds has been extended along with different therapeutic protocols. Here, we monitored the effects of Thymoquinone (TQ) on autophagy via mitochondrial function after modulation of the Wnt/ß-catenin signaling pathway.Human colorectal adenocarcinoma HT-29 cells were treated with TQ (60 µM) and 15 µM Wnt3a inhibitor (LGK974) for 48 h. The survival rate was evaluated using an MTT assay. The expression of Wnt-related factors (c-Myc, and Axin), angiogenesis (VE-Cadherin), and mitophagy-related factors (PINK1, OPTN) was assessed using real-time PCR assay. Protein levels of autophagy factors (Beclin-1, LC3, and P62) were monitored using western blotting. Using flow cytometry analysis, the intracellular accumulation of Rhodamine 123 was evaluated. The migration properties were analyzed using a scratch wound healing assay.Data indicated that TQ can reduce the viability of HT-29 cells compared to the control cells (p < 0.05). The expression of VE-Cadherin was inhibited while the expression of PINK1 was induced in treated cells (p < 0.05). Both LGK974 and TQ-treated cells exhibited activation of autophagy flux (Beclin-1↑, LC3II/I↑, and p62↓) compared to the control group (p < 0.05). TQ can increase intracellular accumulation of Rhodamine 123, indicating the inhibition of efflux mechanisms in cancer cells. Along with these changes, the migration of cells was also reduced (p < 0.05).TQ is a potential phytocompound to alter the dynamic growth of human colorectal HT-29 cells via the modulation of autophagy, and mitophagy-related mechanisms.

Nimbin (N1) and analog N3 from the neem seeds suppress the migration of osteosarcoma MG-63 cells and arrest the cells in a quiescent state mediated via activation of the caspase-modulated apoptotic pathway.

BACKGROUND: Natural products are considered effective sources for new therapeutic research and development. The numerous therapeutic properties of natural substances in traditional medicine compel us to investigate the anti-cancer properties of Nimbin (N1) and its semi-natural analog Nimbic acid (N3) from Azadirachta indica against MG-63 Osteosarcoma cells. MATERIALS AND METHODS: The therapeutic efficacy of N1 and N3 were screened for their toxicity and cytotoxic activity using L6 myotubes, zebrafish larvae and MG-63 osteosarcoma cells. The mitochondrial membrane potential was evaluated using the Rhodamine 123 stain. Further, the nuclear and cellular damage was distinguished using Hoechst and Acridine orange/EtBr stain. The mechanism of cell cycle progression, cellular proliferation and caspase cascade activation was screened using scratch assay, flow cytometry, and mRNA expression analysis. RESULTS: The Nimbin and analogue N3 were found to be non-toxic to normal L6 cells (Rat skeletal muscles), exhibited cytotoxicity in MG-63 cells, and were exposed to be an active inhibitor of cell proliferation and migration. Analogs N1 and N3 induced negative mitochondrial membrane potential when stained with Rhodamine 123, leading to nuclear damage and apoptosis stimulation using AO/EtBr and Hoechst. Further, N1 and N3 induced cell cycle arrest in G0/G1 phase in flow cytometry using PI staining and induced apoptosis by activating the caspase cascade and upregulated Caspase 3 and caspase 9. CONCLUSION: The study demonstrated cytotoxic activity against MG-63 osteosarcoma cells while being non-toxic to normal L6 cells. These compounds inhibited cell proliferation and migration, induced mitochondrial dysfunction, nuclear damage, and apoptosis stimulation. Furthermore, N1 and N3 caused cell cycle arrest and activated the caspase cascade, ultimately leading to apoptosis. These findings indicate that N1 and N3 hold promise as potential candidates used alone or combined with existing drugs for further investigation and development as anti-cancer agents.

[The study of mechanisms of accumulation of daunorubicin and rodamin-123 in cells of human venous blood using cytometry technique].

The article presents comparative data of cytometry estimation of accumulation of daunorubicin and rodamin-123 in cells of peripheralbloodofhealthypeople underincubation ofsubstances in vitro. It is demonstrated that maximal saturation of thrombocytes occurs during the first five minutes, of leukocytes during forty five minutes. The erythrocytes factually never accumulate these compounds. The maximal values of accumulation of substances in leukocytes are characterized by high inter-individual variation. The close correlation (Rs = 0.96-0.98) of parameters of accumulation of substances in lymphocytes and neutrophils testifies the presence ofsimilar mechanisms ofcontrol ofactivity transportation ofxenobiotics in nucleated cells of blood. However, the results of inhibitor analysis of input of Pgp-dependent mechanisms of accumulation of rodamin-123 by leukocytes differ the data received under application of daunorubicin that reflects differences of their intracellular binding sites. The expressed differences between parameters of accumulation ofdaunorubicin and rodamin-123 by leukocytes in various patients determine necessity of individual approach in monitoring of development of medicinal resistance.

From delocalized lipophilic cations to hypoxia: blocking tumor cell mitochondrial function leads to therapeutic gain with glycolytic inhibitors.

An unexpected similarity between cancer and cardiac muscle cells in their sensitivity to anthracyclines and delocalized lipophilic cations (DLC) prompted a series of studies in which it was shown that the positive charge of these compounds is central to their selective accumulation and toxicity in these two distinct cell types. An initial finding to explain this phenomenon was that cancer and cardiac muscle cells exhibit high negative plasma membrane potentials resulting in increased uptake of these agents. However, the p-glycoprotein efflux pump was shown to be another factor underlying differential accumulation of these compounds, since it recognizes positively charged drugs and thereby actively reduces their intracellular concentrations. The delocalized positive charge and lipophilicity of DLCs leads to their retention and inhibition of ATP synthesis in mitochondria. Years later it was realized that cancer cells in the hypoxic portions of solid tumors were similar to those treated with DLCs in relying mainly on anaerobic metabolism for survival and could thus be targeted with a glycolytic inhibitor, 2-deoxy-D-glucose (2-DG). This hypothesis has lead to a Phase I clinical trial in which 2-DG is used to selectively kill the hypoxic tumor cell population which are resistant to standard chemotherapy or radiation.

Photophysical, photochemical, and tumor-selectivity properties of bromine derivatives of rhodamine-123.

The conceptual basis for the development of mitochondrial targeting as a novel therapeutic strategy for both chemotherapy and photochemotherapy of neoplastic diseases rests on the observation that enhanced mitochondrial membrane potential is a common tumor cell phenotype. The potential of this strategy is highlighted by the fact that the toxic effects associated with a number of cationic dyes known to localize in energized cell mitochondria are much more pronounced in tumor cells than in normal cells. Here we evaluate the phototoxic properties of four bromine derivatives of rhodamine-123 toward human uterine sarcoma (MES-SA) and green monkey kidney (CV-1) cells and compare the degrees of tumor cell selectivity associated with these dyes with those associated with two model mitochondrial triarylmethanes (crystal violet and ethyl violet). Selective phototoxicity toward tumor cells was found to be highly dependent upon the lipophilic/hydrophilic character of the cationic photosensitizer. Our experimental data have indicated that the probability of success of mitochondrial targeting in (photo)chemotherapy of neoplastic diseases is higher when the octan-1-ol/water partition coefficient of the drug candidate falls within approximately two orders of magnitude from that of the prototypical mitochondria-specific dye rhodamine-123.

Rhodamine-123: therapy for hormone refractory prostate cancer, a phase I clinical trial.

Rhodamine-123, a lipophilic, cationic, rhodocyanine dye, has been reported to have carcinoma selective toxicity in vitro and in vivo. This phase I clinical trial established the safety and pharmacokinetics of Rhodamine-123 administered to men with hormone refractory prostate cancer. A single dose toxicity study of Rhodamine-123 determined the maximum tolerated dose. A multiple dose toxicity study assessed the safety of Rhodamine-123 at the maximum tolerated dose level. Transient and variable toxicities noted following Rhodamine-123 infusion resolved within 6 hours following infusion. Pharmacokinetic analyses of sera showed no accumulation of drug with repeated monthly administrations. Drug retention was confirmed in prostatic tissue following Rhodamine-123 administration. PSA doubling times lengthened variably suggesting drug efficacy but the data were not statistically significant. The maximum tolerated dose of Rhodamine-123 is 96 mg/m2. The drug can be safely administered at monthly intervals without detectable drug accumulation in serum. Rhodamine-123 is retained by prostatic tumor tissue.

Delocalized lipophilic cations selectively target the mitochondria of carcinoma cells.

Traditional chemotherapies, aimed at DNA replication in rapidly dividing cells, have achieved only limited success in the treatment of carcinomas due largely to their lack of specificity for cells of tumorigenic origin. It is important, therefore, to investigate treatment strategies aimed at novel cellular targets that are sufficiently different between normal cells and cancer cells so as to provide a basis for selective tumor cell killing. Delocalized lipophilic cations (DLCs) are concentrated by cells and into mitochondria in response to negative inside transmembrane potentials. The higher plasma and/or mitochondrial membrane potentials of carcinoma cells compared to normal epithelial cells account for the selective accumulation of DLCs in carcinoma mitochondria. Since most DLCs are toxic to mitochondria at high concentrations, their selective accumulation in carcinoma mitochondria and consequent mitochondrial toxicity provide a basis for selective carcinoma cell killing. Several of these compounds have already displayed some degree of efficacy as chemotherapeutic agents in vitro and in vivo. The effectiveness of DLCs can also be enhanced by their use in photochemotherapy or combination drug therapy. Discovery of the biochemical differences that account for the higher membrane potentials in carcinoma cells is expected to lead to the design of new DLCs targeted specifically to those differences, resulting in even greater selectivity and efficacy for tumor cell killing.

Subcutaneous tumors of mice treated with rhodamine-123 and laser irradiation.

BACKGROUND: Treatment involving photosensitizers and laser irradiation (LIR) in cancer therapy is known as photodynamic therapy (PDT). The purpose of our study was to assess the therapeutic effect of PDT using rhodamine-123 (Rh123) and LIR on subcutaneous tumors (ST) in mice. METHODS: Sarcoma-180 cells (1 x 10(7)) were implanted subcutaneously into the breast area of strain Cr1:CD-1-ICR (BR) female mice. Mice bearing ST were treated with Rh123 or LIR alone, or a combination of both, once a day for 3 successive days. RESULTS: The best therapeutic effect was observed in the group treated with 7.5 mg Rh123 per kilogram of body weight, combined with 75 J/cm2 laser irradiation energy. The group's mortality rate, tumor control rate, mean survival time, and increase in lifespan within 120 days after treatment were 16.7%, 83.3%, 109.4 days, and 135.8%, respectively. The most inhibitory effect on tumor cells was found in the group treated with 15 mg/kg Rh123 and 90 J/cm2 laser irradiation. The biosyntheses of DNA, RNA, and protein in tumor cells of this group was obviously inhibited. CONCLUSION: PDT with the photosensitizer Rh123 and laser irradiation was therapeutically effective in treating subcutaneous tumors of mice. The tumor cells and the syntheses of DNA, RNA, and protein of the tumor cells in these PDT treated mice were obviously inhibited.

The effect of rhodamine-123 on 3 prostate tumors from the rat.

PURPOSE: Rhodamine-123 (Rh-123) was found to have a specific attraction to the mitochondria of tumor cells. The destruction of rat prostate tumor cells by Rh-123 is described. MATERIALS AND METHODS: Tissue was used from rat prostate studies of Rh-123 treatment of R3327-H, PA III prostate tumor of Pollard and the autochthonous tumor in Lobund-Wistar rats. All tissues were fixed in 10% buffered formalin, paraffin embedded and sectioned at 1 to 3 micron. for good cellular detail. RESULTS: Destructive processes were seen in all 3 rat prostate tumor models evaluated. The changes included acinar cell clumping, acinar destruction with scarring, cyst formation within acinar cells and increased stromal cells. CONCLUSIONS: Since all tumor models were found to respond to Rh-123 in a similar manner, any of them could be used for the evaluation of anticancer agents. These studies demonstrated that Rh-123 was effective in suppressing the growth of hormone sensitive and insensitive rat prostate tumor cells.