Enhancement of Apoptotic and Autophagic Induction by a Novel Synthetic C-1 Analogue of 7-deoxypancratistatin in Human Breast Adenocarcinoma and Neuroblastoma Cells with Tamoxifen

The overall goal of this procedure is to evaluate the selective toxicity and mechanism of cell death induced by J CTH four, A synthetic analog of the natural compound pancreat statin alone and in combination with tamoxifen in human breast cancer and neuroblastoma cells. This is accomplished by first culturing mc seven breast cancer cells, SHSY five Y neuroblastoma cells and normal fetal fibroblasts or NFF. The second step is to evaluate the cytotoxicity of J CTH four and Tamoxifen alone or in combination.

By treating cells with these drugs and monitoring time dependent morphological changes characteristic of apoptosis and autophagy. Next, the mechanism of induction of apoptosis and autophagy is investigated by monitoring mitochondrial membrane potential and reactive oxygen species production in isolated mitochondria from these cancer cells. Ultimately time lapse photography and various biochemical assays are used to demonstrate that JCT four induces apoptosis and autophagy in human breast cancer and neuroblastoma cells, and these effects are dramatically enhanced by the addition of tamoxifen.

Importantly, these treatments do not affect the survival of non-cancerous human fibroblasts, indicating that J CTH four in combination with tamoxifen could be used as a safe and very potent anti-cancer therapy against breast cancer and neuroblastoma cells. The implications of this technique extend toward preclinical evaluation and development of combinatorial therapy for cancer because one can monitor the efficacy and mode of cancer cell deaths induced by potential anti-cancer compounds. Though this methodic can provide insight into the mechanism of cytotoxicity induced by J CT four and tamoxifen and breast cancer and neuroblastoma cells, it can also be applied to other anti-cancer systems and other potential anti-cancer compounds.

We first had the idea of this method when we observed synergistic action of j ct H four and Tamoxifen in killing cancer cells. Tamoxifen generally activates autophagy and J cts four induces apoptotic cell death in cancer cells. Therefore, we wanted to monitor the morphology of cancer cells undergoing apoptosis or autophagy.

MC seven human breast adenocarcinoma cells are grown in RPMI 1640 medium and maintained at 37 degrees Celsius and 5%carbon dioxide. It is important to work under sterile conditions in a class two biosafety cabinet when preparing these cells for drug treatment and time-lapse microscopy plate approximately 2.0 times 10 to the fifth cells in a 35 millimeter glass bottom culture dish and incubate at 37 degrees Celsius and 5%carbon dioxide. When cells reach 60 to 70%confluence, they're ready to be treated with the synthetic pancreas statin analog j, CT H four, or Tamoxifen or tam at these concentrations, one micromolar of J CT four and 10 micromolar of tam.

The three experimental conditions are treatment with J CT four alone treatment with TAM alone and treatment with a combination of JCTH four and TAM DMSO alone is added to the untreated control. After adding the appropriate agent or agents return the cells to the incubator after treatment of cells for 48 hours, place cells in a heated chamber at 37 degrees Celsius with 5%carbon dioxide on a stage of a Leica DMI 6, 000 fluorescent microscope. Using LAS AF 6, 000 software set the microscope to take phase contrast or brightfield micrographs at 400 times magnification every five minutes for 18 hours.

To begin this procedure, trypsin is a confluent T 75 flask of C seven SHSY five Y or normal human fetal fibroblast cells. After the cells have been lifted at five to 10 milliliters of media to the trypsin and place the cell suspension in a 15 milliliter conical tube centrifuge the cells at 600 times G for five minutes. Return the tube to the biosafety cabinet, remove the supernatant and resus, suspend the cells in one to three milliliters of media depending on the size of the cell pellet place 10 microliters of cell suspension in a fuge tube, add 10 microliters of trian blue dye and mix with a micro pipette.

Next load 10 microliters of the trian blue suspended cells on a hemo cytometer. Count the cells and calculate the cell concentration of the cell suspension in the original 15 milliliter conical tube. Based on the calculations, use an appropriate volume of original cell suspension to create diluted cell suspensions with the following concentrations.

1.5 times 10 to the fifth cells per milliliter for MCF seven and SHSY five Y cells and 5.0 times 10 to the fourth cells per milliliter. For human fetal fibroblast cells transfer 100 microliters of the diluted cell suspensions to each well of a 96 well clear bottom tissue culture plate. This will result in 15 times 10 to the third MCF seven or S sh SY five Y cells per well, or 5.0 times 10 to the third NFF cells per well.

Blanks of 100 microliters of media without cells must be plated as well. Incubate the cells at 37 degrees Celsius in 5%carbon dioxide overnight on the following morning. Treat the cells with one micromolar JCT four and 10 micromolar tam both alone and in combination.

Incubate MCF seven and NF cells for 72 hours and S SH SY five Y cells for 48 hours. After treatment and incubation with drugs, add 10 microliters of water soluble tetra oleum salt or wst one reagent to each well and incubate the plates for four hours at 37 degrees Celsius and 5%carbon dioxide prior to starting mitochondrial isolation. Prepare about 10 milliliters each of hypotonic buffer and reaction buffer and keep both solutions on ice.

Trypsin eyes about eight to 10 cofluent T 75 tissue culture flasks of SHSY five Y cells. After adding media to neutralize the trypsin, collect the cell suspensions in 50 milliliter conical tubes and centrifuge the tubes at 600 times G for five minutes at four degrees Celsius. Remove the supernatant and resuspend cell pellets in about 10 to 20 milliliters of cold PBS centrifuge again at 600 times G for five minutes at four degrees Celsius.

Repeat this wash process once after the final centrifugation. Remove the supernatant and resuspend cells in cold hypotonic. Buffer homogenized cells manually with a glass tissue grinder.

Centrifuge the cell lysate at 600 times G for five minutes at four degrees celsius and transfer the supernatant to a new tube. Finally, centrifuge the resultant supernatant at 15, 000 times G for 15 minutes at four degrees Celsius. Remove the cytosolic supernatant and resuspend the mitochondrial pellet in cold reaction buffer.

After isolating mitochondria from cells, the protein concentration of the sample of isolated mitochondria is estimated using a standard curve of known concentrations of one milligram per milliliter BSA with the BioRad protein assay. Using the estimated concentration of protein of the isolated mitochondria solution, calculate the volume of the solution to give 20 micrograms of protein. Pipette this volume into each well of an opaque 96 well plate to load 20 micrograms of protein per well fill each well in the plate to a total volume of 100 microliters with the following reaction.

Buffer drunk treatment or controls plex red reagent and horse radish peroxidase. Then take fluorescence readings on a spectro fluorimeter natural pancreas. Statin or PST has been shown to selectively induce apoptosis in various cancer cells due to the low availability of PST analogs of seven deoxy pancreas statin were synthesized a screen of these analogs for similar anti-cancer activity in seven human breast adenocarcinoma cells and SHSY five Y neuroblastoma cells identified a C one acid oxy methyl derivative J CT H acetate four or J CT four.

In the first phase of experiments, morphological changes over time in C seven cells following treatment with JC TH four and TAM alone and in combination were monitored by time-lapse microscopy. This first video of phase contrast pictures is a control in which C seven cells were treated with solvent for 48 hours and then monitored for 18 hours as seen here. These cells exhibited no major changes in morphology.

In contrast, after 48 hours of one micromolar J CT H four treatment MCF seven cells exhibited morphological changes associated with apoptosis such as shrinkage, blobbing, and apoptotic body formation. On the other hand, TAM treatment alone in C seven cells produced a very distinct morphology including punctate inclusions indicative of autophagosome associated with autophagy. Very minimal apoptotic morphology was observed and cells generally exhibited a healthy morphology comparable to that of the solvent control treated MC seven cells.

Interestingly in the presence of tam, the ATO induction by J CT H four was drastically enhanced as indicated by increased ATO morphology in MCF seven cells after 48 hours, which is illustrated in this video. In the second phase of experiments, fluorescent dyes were used to evaluate the induction of apoptosis after one micromolar J cth four treatment for 72 hours in mc seven cells and 48 hours in hs. Y five Y cells die was used to monitor nuclear morphology.

Results indicated condensed brightly stained nuclei accompanied by apoptotic bodies in MC seven and HS Y five Y cells indicative of ATO induction. TAM treatment alone yielded minimal ATO NU nuclear morphology in C seven and hs. Y five Y cells Nuclei were large round and dimly stained with hooks to die comparable to the solvent control group.

In agreement with what was observed from time lapse microscopy nuclei of MCF seven and SH SY five Y cells displayed a market increase in apoptotic morphology with the combination treatment. After 72 hours to verify ATO induction cells were evaluated for phosphatidyl Syrian externalization a marker for apoptosis via an nexin five binding assay MCF seven and SSH SY five Y cells treated for 72 and 48 hours respectively with one micromolar J CT four alone and in combination with 10 micromolar TAM were positive for a nexin five binding indicated by the green fluorescence confirming the induction of apoptosis. No evident externalization of phosphatidyl seine was observed in mc seven and S hs Y five Y cells treated with Tam Malone.

Importantly, externalization of phosphatidyl Seine was also not observed in NFF cells treated with J CT H four and TAM alone or in combination after 72 hours. Therefore, J CT four alone and in combination with TAM selectively induces apoptosis in MCF seven and s sh SY five Y cells without affecting the survival of non-cancerous human fibroblasts. To quantify the effect of JC TH four alone and in combination with tam, a water-soluble tetra oleum salt or wst one based colormetric assay for cell viability, an indicator of active cell metabolism was performed on C seven cells treated for 72 hours and HSY five Y cells treated for 48 hours compared to the solvent control one.

Micromolar JCTH four decreased active cell metabolism by over 50%while 10 micromolar TAM alone exhibited no significant difference in both MCF seven and HS Y five Y cells. Interestingly, in C seven and SHS Y five Y cells, the addition of TAM to J CT H four insult resulted in a synergistic decrease in cell metabolism. In contrast to C seven and SHSY five Y cells.

NFF cells were drastically less sensitive to both J CTH four alone and J CT four with TAM as shown by these results. Hence, J CTH four demonstrates selective synergistic activity with TAM in C seven and s SH SY five Y cells to see if J CT H four is targeting the mitochondria to induce apoptosis. Mitochondrial membrane potential in whole cells was monitored.

MC seven cells were treated for 72 hours and stained with tetraethyl rumine methyl ester or TMRM as shown in this figure one micromolar JC TH four decreased mitochondrial membrane potential indicated by the loss of red fluorescence. However, with the addition of 10 micromolar tam, a greater dissipation of mm MP was observed while 10 micromolar tam alone had no evident effect on mitochondrial membrane potential as increases in reactive oxygen species or ROS generation have been associated with mitochondrial membrane permeation and apoptosis induction. The production of ROS was assessed with plex red dye in isolated mitochondria from SHSY five Y cells treated with one micromolar J CTH four and 10 micromolar TAM alone or in combination.

The results obtained are shown in this graph with fluorescence readings expressed as relative fluorescence units or RFU increases in ROS generation were observed with j ct H four and Tamal alone. Interestingly, combination treatment yielded a greater increase in ROS production, a well-known inducer of ROS production in mitochondria. Paraquat, or PQ was utilized as a positive control since autophagic induction has been associated with chemotherapeutic insult by many different compounds.

The induction of autophagy was evaluated. MONOBENZYL cadaver or MDC staining was performed on MCF seven cells treated with one micromolar J CT H four and 10 micromolar TAM alone and in combination for 72 hours. Blue punctate fluorescence indicative of autophagosome was present in C seven cells treated with J CTH four and TAM alone and in combination.

Notably the intensity of MDC staining was the greatest with the combination treatment followed by TAM alone and J CTH four alone during autophagy, micro tubular associated protein one, light chain three or lc three one normally situated in the cytosol is lipid dated with phosphatidyl ethanolamine to lc three, two, and subsequently localized to the autosomal membranes to verify the induction of autophagy levels of lc three two were assessed in C seven cells treated for 72 hours via western blot analyses. DTO metric analyses of lc three two to lc three one ratios revealed that one micromolar JC TH four slightly induced the conversion of lc three one to LC three two, while 10 micromolar TAM produced a greater autophagic response. Significantly, the combination treatment resulted in the greatest induction of autophagy yielding an lc three, two to lc three, one ratio greater than three.

The results from our experiments indicate that treatment of human breast cancer and neuroblastoma cells with a combination of JCTH four and Tamoxifen drastically enhances apoptotic and autophagic induction by mitochondrial targeting with minimal effect on non-cancerous fibroblast. It is an exciting possibility that this combinatorial treatment could be used as a safe and very potent anti-cancer therapy. The findings of this work paved the way for the further development of a novel non-toxic chemotherapy for cancer by combinatorial treatment with Tamoxifen and JCTs four independent of estrogen receptor.

After watching this video, should have a good understanding of how to monitor morphological changes in cells undergoing apoptosis and autophagy. Furthermore, we can use these methodologies for screening new potential anti-cancer agents and evaluating their effects on mitochondrial permeability.