|Ahead of print publication
Increased breast cancer cell sensitivity to cisplatin using a novel small molecule inhibitor
Eleana Hatzidaki1, Vasiliki Daikopoulou1, Panagiotis Apostolou1, Dimitrios Athanasios Ntanovasilis1, Ioannis Papasotiriou2
1 Research Genetic Cancer Centre SA, Florina, Greece
2 Research Genetic Cancer Centre GmbH, Zug, Switzerland
|Date of Submission||30-Aug-2019|
|Date of Decision||01-Nov-2019|
|Date of Acceptance||07-Jan-2020|
|Date of Web Publication||19-Oct-2020|
RGCC International GmbH, Baarerstrasse 95, Zug
Source of Support: None, Conflict of Interest: None
Background: Although cisplatin is used for the treatment of more than half of cancer patients, its use is restricted by serious side effects as well as the development of cisplatin-resistant cancer cells, limiting its use. In RGCC we have synthesized an intermediate molecule in an ERK inhibitor synthesis process.
Aims and Objectives: The aim of the study was to evaluate the effects of combined cisplatin plus RGCC molecule treatment on MCF-7 and MDAMB231 breast cancer cell viability, proliferation, ability to form clones and migrate, as well as the effects on cell cycle and gene expression.
Materials and Methods: Cell viability and proliferation were measured by Crystal violet exclusion dye and MTT respectively. Clone formation and wound healing assays were also used for clone formation and cell migration evaluation. Cell cycle was studied by flow cytometry, expression of genes was evaluated by PCR and protein expression was evaluated by western blot.
Results: It was found that combination therapy decreased cell viability and proliferation, caused growth arrest, decreased cancer cell invasiveness and the ability to form clones as well as perturbed the expression of genes involved in ERK, cell cycle and cell death pathways.
Conclusion: Although the exact mechanism of action of the combination therapy remains to be investigated, it was found that it is more effective than cisplatin monotherapy. Our findings could potentially lead to a new therapeutic regime for the treatment of cancer.
Keywords: Breast cancer, cisplatin, growth arrest, proliferation, Research Genetic Cancer Centre, viability
|How to cite this URL:|
Hatzidaki E, Daikopoulou V, Apostolou P, Ntanovasilis DA, Papasotiriou I. Increased breast cancer cell sensitivity to cisplatin using a novel small molecule inhibitor. J Can Res Ther [Epub ahead of print] [cited 2020 Dec 3]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=298619
| > Introduction|| |
Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer-related death in women. A percentage of breast cancers are hormone sensitive and can be treated by anti-estrogen drugs. Another percentage is human epidermal growth factor receptor (HER-2) positive, so those women can benefit from treatments like Herceptin. However, about 15%–20% of breast cancer tumors have neither estrogen nor progesterone receptors and also do not overexpress HER2. Since these so-called triple-negative tumors (triple negative breast cancer [TNBC]) are treatable with neither Herceptin nor antiestrogen drugs, the prognosis for TNBC patients has been poor. In those patients, chemotherapy is administered. There is no clear consensus about the therapeutic regiment to be followed, but cisplatin can be given either alone or in combination in TNBC. Findings suggest that neoadjuvant use of cisplatin results in high rates of complete pathological response in patients with TNBC. In the first-line setting, where cisplatin can be the most active, other less toxic regimens that have similar or better activity are favored.
Cisplatin is considered to be a success story in cancer treatment. It is a very potent chemotherapeutic drug that can be used for the management of a wide range of cancer types including head and neck, ovarian, breast, leukemia, kidney, and testicular cancer., In fact, it is so effective that it is used in over half of all cancer patients. Cisplatin mode of action relies on the construction of both intra- and inter-strand DNA crosslinks, mainly with purine bases such as adenine and guanine and also glutathione, inhibiting DNA replication. Since cisplatin's mode of action is considered to be due to formation of adducts with DNA, one would assume that its action will be clinically manifested when the cell divides and the DNA strands come apart and become available for pairing with the drug. In this way, cells which are replicating faster will also become affected faster. However, only a small percentage of cisplatin accounting for 1% actually interacts with DNA. Therefore, interference with DNA replication cannot be the only mechanism of its antitumor activity. In fact, an effect in cell proliferation can be seen in as early as 6 h from treatment, indicating the presence of other mechanisms that can be activated and promote cytotoxicity.
Despite its success, there are several disadvantages with the use of cisplatin in cancer management. These include severe side effects which cause dose-limiting toxicity thus restricting its application. The development of cisplatin-resistant cancer cells is another challenge. The majority of patients will relapse and become refractory to cisplatin at some point. Those resistance mechanisms are multifactorial and can be due to a number of mechanisms including changes in drug uptake, efflux, deactivation, DNA repair, apoptosis pathways, and other signaling molecules involved in various pathways such as JAK, STAT, PI3K, Ras, or EGF to name a few., To combat cisplatin resistance, a number of combination therapies have been developed. It is not uncommon for cisplatin to be administered along with one, two, three, or even four additional drugs.
In Research Genetic Cancer Centre (RGCC), we are in the process of synthesizing a novel Extracellular signal-regulated kinase (ERK) inhibitor. Currently, we are testing an intermediate molecule that was shown to decrease breast cancer cell proliferation.
The aim of the present study was the evaluation of the effects of combined treatment of cisplatin plus RGCC small molecule on MCF-7 and MDAMB231 breast cancer viability, proliferation, ability to form clones and migrate, as well as the effects on cell cycle and gene expression. The results from the combined treatment were compared with those obtained using monotherapy. MCF-7 is a model of hormone-sensitive breast cancer, and MDAMB231 represents TNBC.
The ultimate goal is to find a treatment regimen that is less toxic, does not induce resistance, and could be potentially more effective in treating cancer.
| > Materials and Methods|| |
Cell lines and treatments
MCF-7 and MDAMB-231 cell lines were obtained from American Type Culture Collection (Manassas, VA, USA). They were grown at 37°C and 5% CO2 and maintained in RPMI medium (Sigma-Aldrich, St. Louis, MO, USA). The culture media were enriched with 10% fetal bovine serum (Life Technologies, Carlsbad, CA, USA), 2 mM L-glutamine (Sigma-Aldrich, St. Louis, MO, USA) and 1% nonessential amino acids (Sigma-Aldrich, St. Louis, MO, USA) for MCF-7 cells and 15% fetal bovine serum and 2 mM L-glutamine for MDAMB231. Both cells were grown until 80%–90% confluency before experiments. Cells were treated with various concentrations of cisplatin (Sigma-Aldrich, St. Louis, MO, USA) according to each experiment, in the presence or absence of 30 μM RGCC small molecule. RGCC small molecule was also studied alone. RGCC small molecule is an intermediate product in ERK protein inhibitor synthesis. Its nature cannot be disclosed yet because of patent pending. 30 μM RGCC small molecule was chosen based on previous published studies. For proliferation and viability, a range of cisplatin concentrations were used. For further experiments, we chose to use cisplatin concentrations that alone had no effect of either viability or proliferation.
The effect of cisplatin with and without RGCC small molecule on cell viability was measured using crystal violet inclusion assay. Briefly, 20 × 103 cells, both MCF-7 and MDAMB231, were plated in 96 well plates and left overnight for adhesion. Next day cells were incubated for 24 h with various cisplatin concentrations (0.1, 1, 10, 100, 150 μM with or without 30 μM RGCC small molecule. Same volume dimethyl sulfoxide (DMSO) was used as control. After the incubation period, supernatants were removed and 10 μl formalin 10% was added to each well for 20 min at room temperature. After cell fixation, formalin was removed and 100 μl0.25% aqueous crystal violet was added to each well and left for 10 min. Plates were washed with 100 μlwater for injection 3 times. Finally, 100 μl of 33% glacial acetic acid was added to each well for dye solubilization. Absorbance was read at 570 nm using a uQuant spectrophotometer (MQX200, BIOTEK, Winooski, Vermont, USA).
The effect of cisplatin with and without RGCC small molecule on cell proliferation was measured using the MTT dye assay. Briefly, 20 × 103 cells, both MCF-7 and MDAMB231, were plated in 96 well plates and left overnight for adhesion. Next day cells were incubated for 24 h with various cisplatin concentrations (0.1, 1, 10, 100, 150 μM) with or without 30 μM RGCC small molecule. Same volume DMSO was used as control. After the incubation period, 20 μl MTT (5 mg/ml) was added per well and the plate was left for 3 h at 37°C, 5% CO2. After the end of the incubation period, supernatants were carefully removed and 100 μl of DMSO was added in each well. Plates were left in the incubator for 5 min for formazan crystal solubilization. Absorbance was read at 595 nm using a uQuant spectrophotometer (MQX200, BIOTEK, Winooski, Vermont, USA).
The effect of cisplatin with and without RGCC small molecule on the ability of a cell to form a colony was measured using clone formation assay. Briefly, 100 cells, both MCF-7 and MDAMB231 were plated in 6 well plates with 0.1 μM and 1 μM cisplatin, with and without 30 μM RGCC small molecule. Cells were then incubated at 5% CO2, 37°C until colony formation. Colonies were fixed with methanol and stained with 1% crystal violet. % difference in colony formation compared to control was then calculated using the ColonyArea plugin in ImageJ (NIH, LOCI, University of Wisconsin, Wisconsin, USA).
Wound healing assay
The effect of cisplatin with and without RGCC small molecule on cell migration was measured using the wound healing assay. Briefly, 2 × 105 cells, both MCF-7 and MDAMB231 were plated in 6 well plates and left to grow at 37°C, 5% CO2. When confluency was reached, cells were scratched with a yellow tip and treated with 0.1 μM and 1 μM cisplatin with or without 30 μM RGCC small molecule. Three different areas along the scratch were marked and captured before and after treatment and % difference in migration was calculated using Wound Healing Tool plugin in ImageJ.
Cell cycle analysis
The effect of cisplatin with and without RGCC small molecule on cell cycle was analyzed using flow cytometry. Briefly, cells, both MCF-7 and MDAMB231 were plated in T25 tissue culture plates and when 80% confluent, treated with 0.1 μM and 1 μM cisplatin with or without 30 μM RGCC small molecule. Cells were incubated for 24 h at 5% CO2, 37°C. After the end of the incubation period, cells were detached using trypsin, washed with 2 ml phosphate-buffered saline (PBS) by centrifugation 5 min, 500 g and the supernatant was discarder. Cell pellet was fixed with the addition of 2 ml ice cold methanol. Cells were then centrifuges for 5 min, 800 g and washed with PBS by centrifugation 5 min, 500 g. Supernatant was discarded and cells were re-suspended in 500 μl propidium iodine (PI) staining buffer (BD Biosciences, Franklin Lakes, NJ, USA). Cells were incubated with PI buffer for 15 min and then analyzed in a Cytomics FC500 (Beckman Coulter, Brea, CA, USA).
The effect of cisplatin with and without RGCC small molecule on gene expression was also studied. Genes that were studied were involved in ERK pathway such as ERK1, ERK2, KRAS, ARAF, BRAF, CRAF or cell death and cell cycle such as BAX (apoptotic), BCL2 (anti-apoptotic), CDKN1A, and CDKN1B. Total RNA from cultured cells was extracted using an RNeasy Mini Kit (Qiagen, Hilden, Germany). Total RNA samples were evaluated spectrophotometrically, and 1 μg of each RNA sample was used as a template for cDNA synthesis using a PrimeScript RT Reagent Kit (Takara, Beijing, China). Real-time quantitative polymerase chain reaction (qPCR) was then performed using KAPA SYBR Fast Master Mix (2X) Universal (KAPA Biosystems, MA, USA) in a final volume of 20 μl. Specific primers were designed using Beacon Designer. Primer sequences were evaluated by BLAST searching to exclude those that would amplify undesired genes. The PCR program was as follows: initial denaturation at 95°C for 2 min followed by 45 cycles of denaturation at 95°C for 10 s and annealing at 59°C for 30 s. Melting-curve analysis was performed from 65°C to 95°C with 0.5°C increments for 5 s at each step. The qPCR products were run on agarose gels and visualized, to validate the results. ΔCt value was used for analysis of experiments. In all sets of reactions, cDNA from Universal Human Reference RNA (Agilent, CA, USA) was used as a positive control. Template-free and negative controls were also used in all experiments. All the reactions were performed in triplicate. ΔCt value was used for analysis of experiments. Finally, relative quantification was performed using the normal samples as the reference group according to Livak and Schmittgen.
The effect of cisplatin with and without RGCC small molecule on ERK and phospho-ERK protein levels was studied by western blot. MCF-7 and MDAMB231 cells were grown until 80% confluency in T25 tissue culture plates. Cells were then treated with 0.1 μM and 1 μM cisplatin with or without 30 μM RGCC small molecule. Cells were harvested using ice cold RIPA buffer ([Sigma-Aldrich, St. Louis, MO, USA] supplemented with protease and phosphatase inhibitor cocktails [Sigma-Aldrich, St. Louis, MO, USA]). Cells were vortexed briefly and incubated on ice for 30 min. Whole cell lysate was collected by spin at 12,000 g for 10 min at 4°C. Samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis using a Mini Protean Tetra System (BioRad, Hercules, CA, USA) and transferred onto nitrocellulose membranes using a Trans-blot SD, Semi-dry Transfer cell (BioRad, Hercules, CA, USA). Transferred membranes were blocked by 1 h incubation with 5% bovine serum albumin in tris buffered saline (TBS)/Tween-20 and incubated with the respective primary antibody in blocking buffer at 4°C, overnight (anti-total ERK1/2 Ab, Novus Biologicals, Brianwood Ave, CO, USA; anti-Phospho ERK1/2, Antibodies Online, Aachen, Germany; anti-actin, Biolegend, San Diego, CA, USA). Membranes were washed with TBS/Tween-20 3 times for 10 min and incubated with the respective secondary antibodies for 1 h (Goat Anti-Rabbit IgG Antibody, Alkaline Phosphatase conjugate, Millipore, Burlington, MA, USA). Protein signals were detected using BCIP/NBT solution (Sigma-Aldrich, St. Louis, MO, USA) and captured by a GelDocXR+ (BioRad, Hercules, CA, USA). Equal loading was confirmed by actin.
Data are expressed as mean ± standard error of the mean. Unless stated otherwise, statistical significance was determined using Student's t-test and statistical significance was achieved when P < 0.05.
| > Results|| |
RGCC small molecule further decreases MCF-7 cell viability
First, a wide range of cisplatin concentrations in the presence or absence of 30 μM RGCC small molecule was tested on cell viability. The aim was to test both MCF-7 and MDAMB231 sensitivity. In both cells, 0.1 μM and 1 μM cisplatin do not decrease viability. Significant decrease in both viabilities is noticed at 10 μM cisplatin and higher which are usually used in the clinical setting. However, when RGCC is also added, there is an additional significant decrease in viability both at 0.1 μM and 1 μM cisplatin in MCF-7 [Figure 1]a. In MDAMB231 cells RGCC addition does not cause any additional statistical difference except at 10 μM [Figure 1]b. Therefore, in MCF-7 RGCC addition sensitizes the cells to cisplatin causing cell death as lower cisplatin concentrations. In MBA-MB-231, RGCC addition does not have any additive effect and the decrease in viability is equal to the one using cisplatin only. Therefore, the addition of RGCC small molecule sensitized MCF-7 cells to cisplatin causing a further decrease in viability.
|Figure 1: (a) The effect of various concentrations of cisplatin with and without 30 μM RGCC small molecule in MCF-7 viability as measured by crystal violet inclusion dye. *Significance compared to control (10 μM cisplatin P = 4.7 × 10−6, 100 μM cisplatin P = 3.2 × 10−18, 150 μM cisplatin P = 4.2 × 10−13, 0.1 μM cisplatin + RGCC P = 0.01, 1 μM cisplatin + RGCC P = 0.002, 10 μM cisplatin + RGCC P = 1.9 × 10−8, 100 μM cisplatin + RGCC P = 1.7 × 10−20, 150 μM cisplatin + RGCC P = 1.9 × 10−20). #Significance between same cisplatin concentration with and without RGCC (0.1 μM ± RGCC P = 0.002, 1 μM ± RGCC P = 0.006, 150 μM ± RGCC P = 0.04) (b) The effect of various concentrations of cisplatin with and without 30 μM RGCC small molecule in MDAMB231 viability as measured by crystal violet inclusion dye. *Significance compared to control (10 μM cisplatin P = 1.2 × 10−9, 100 μM cisplatin P = 6 × 10−20, 150 μM cisplatin P = 2.3 × 10−20, 0.1 μM cisplatin + RGCC P = 0.003, 1 μM cisplatin + RGCC P = 0.02, 10 μM cisplatin + RGCC P = 0.0002, 100 μM cisplatin + RGCC P = 5.2 × 10−19, 150 μM cisplatin + RGCC P = 2.6 × 10−18). #Significance between same cisplatin concentration with and without RGCC (10 μM ± RGCC P = 0.0006) Results represent mean ± standard error of the mean|
Click here to view
RGCC small molecule further decreases MCF-7 and MDAMB231 proliferation
The same range of cisplatin concentrations in the presence or absence of 30 μM RGCC small molecule was tested on cell proliferation as well. In MCF-7 there was a significant decrease in proliferation at 1 μM cisplatin and higher, however, when RGCC is added, cell proliferation decreased at 0.1 μM cisplatin. RGCC alone also caused a significant decrease in proliferation [Figure 2]a. In MBAMB231, neither 0.1 μM nor 1 μM have any effect. Decrease in proliferation is noted from 10 μM and higher. However, there is a significant decrease even at 0.1 μM cisplatin when RGCC is added [Figure 2]b. Therefore, the addition of RGCC small molecule sensitized both MCF-7 and MDAMB231 cells to cisplatin causing a further decrease in proliferation.
|Figure 2: (a) The effect of various concentrations of cisplatin with and without 30 μM RGCC small molecule in MCF-7 proliferation as measured by MTT. *Significance compared to control (1 μM cisplatin P = 0.004, 10 μM cisplatin P = 9.4 × 10−24, 100 μM cisplatin P = 1.4 × 10−51, 150 μM cisplatin P = 1.9 × 10−30, RGCC P = 3.9 × 10−30, 0.1 μM cisplatin + RGCC P = 0.006, 1 μM cisplatin + RGCC P = 4.3 × 10−6, 10 μM cisplatin + RGCC P = 2.4 × 10−14, 100 μM cisplatin + RGCC P = 6.9 × 10−51, 150 μM cisplatin + RGCC P = 8.8 × 10−53). #Significance between same cisplatin concentration with and without RGCC (0.1 μM ± RGCC P = 5.7 × 10−23, 1 μM ± RGCC P = 1.1 × 10−27, 10 μM ± RGCC P = 1 × 10−10, 100 μM ± RGCC P = 2.2 × 10−10, 150 μM ± RGCC P = 1.7 × 10−6) (b) The effect of various concentrations of cisplatin with and without 30 μM RGCC small molecule in MDAMB231 proliferation as measured by MTT. *Significance compared to control (10 μM cisplatin P = 2.4 × 10−8, 100 μM cisplatin P = 2.2 × 10−27, 150 μM cisplatin P = 4.9 × 10−27, 0.1 μM cisplatin + RGCC P = 2.6 × 10−11, 1 μM cisplatin + RGCC P = 1.8 × 10−17, 10 μM cisplatin + RGCC P = 3.1 × 10−23, 100 μM cisplatin + RGCC P = 3.3 × 10−34, 150 μM cisplatin + RGCC P = 3.7 × 10−43). # Significance between same cisplatin concentration with and without RGCC (0.1 μM ± RGCC P = 5.5 × 10−9, 1 μM ± RGCC P = 2.6 × 10−10, 100 μM ± RGCC P = 0.01) Results represent mean ± standard error of the mean|
Click here to view
RGCC small molecule further decreases MCF-7 and MDAMB231 clone formation
Based on the previous results, we decided to treat cells with 0.1 μM and 1 μM cisplatin because in these concentrations there was no effect on cells, but when RGCC was added, there was a significant decrease in both viability and proliferation. The ability of a single cell to form clones was studied as well. It was found that in MCF-7 cells even a low concentration of 0.1 μM cisplatin can decrease clone formation. RGCC has the same effect. However, when used in combination, 0.1 μM cisplatin and RGCC, clone formation was decreased significantly compared to 0.1 μM cisplatin alone. For 1 μM cisplatin there is no significant difference, since clone formation was completely inhibited [Figure 3]a. In MDAMB231 cells, 0.1 μM cisplatin and RGCC do not affect clone formation significantly. 1 μM cisplatin decreases the formation of clones and when used in combination with RGCC, the decrease is more evident [Figure 3]b. Therefore, the addition of RGCC small molecule sensitized both MCF-7 and MDAMB231 cells to cisplatin causing a further decrease in clone formation.
|Figure 3: (a) The effect of 0.1 μM and 1 μM cisplatin with and without 30 μM RGCC in MCF-7 clone formation. *Significance compared to control (0.1 μM cisplatin P = 0.005, 1 μM cisplatin P = 4.2 × 10−15, 30 μM RGCC P = 1.1 × 10−15, 0.1 μM cisplatin + RGCC P = 2.4 × 10−9, 1 μM cisplatin + RGCC P = 7.7 × 10−11). #Significance between same cisplatin concentration with and without RGCC (0.1 μM cisplatin ± RGCC P = 0.045). (b) The effect of 0.1 μM and 1 μM cisplatin with and without 30 μM RGCC in MDAMB231 clone formation. *Significance compared to control (1 μM cisplatin P = 2.9 × 10−7, 1 μM cisplatin + RGCC P = 1.2 × 10−9). #Significance between same cisplatin concentration with and without RGCC (1 μM cisplatin ± RGCC P = 0.02)|
Click here to view
RGCC small molecule further decreases MCF-7 migration
Next, cell migration was studied using the wound assay. Cisplatin alone did not cause any effects. RGCC alone decreased MCF-7 migration significantly. RGCC addition decreased migration when used in combination with 1 μM cisplatin compared to control. There was a very strong tendency for decrease in migration when RGCC plus 1 μM cisplatin was used compared to cisplatin alone [Figure 4]a. In MDAMB2131 RGCC alone decreased migration. All the other treatments did not produce a statistically significant result, due to the great variability of the results [Figure 4]b. Therefore, the addition of RGCC small molecule had no effect on either cell migration. There was a tendency for MCF-7 cells to further decrease migration at 1 μM plus RGCC but it was not statistically significant.
|Figure 4: (a) The effect of 0.1 μM and 1 μM cisplatin with and without 30 μM RGCC in MCF-7 migration. *Significance compared to control (RGCC P = 0.01, 1 μM cisplatin + RGCC P = 0.006). (b) The effect of 0.1 μM and 1 μM cisplatin with and without 30 μM RGCC in MDAMB231 migration. *Significance compared to control (RGCC P = 0.0008)|
Click here to view
RGCC small molecule alters cell cycle in MCF-7 and MDAMB231
The effect of cisplatin and RGCC small molecule on cell cycle was determined by flow cytometry. From the results in can be seen that in MCF-7 0.1 μM and 1 μM cisplatin decreased G1 and increased S and G2 denoting proliferation. However, when RGCC was added at 0.1 μM cisplatin, G1 was increased and S was decreased. Similarly, at 1 μM, RGCC decreased S and G2 and increased G1 [Figure 5]a. Similar results can be seen in MBAMB231 cells. 0.1 μM and 1 μM cisplatin decreased G1 and increased S and G2. RGCC addition had no effect when added at 0.1 μM treated cells, but at 1 μM G1 and S increased and G2 decreased denoting growth arrest [Figure 5]b. Therefore, the addition of RGCC in both cell lines increased their sensitivity to cisplatin causing growth arrest, with MCF-7 being more sensitive than MBAMB231.
|Figure 5: (a) The effect of 0.1 μM and 1 μM cisplatin with and without 30 μM RGCC in MCF-7 cell cycle. Compared to control: G1: 0.1 μM cisplatin P = 0.01, 1 μM cisplatin P = 0.0003, 0.1 μM cisplatin + RGCC P = 0.01, 1 μM cisplatin + RGCC P = 0.01. S: 1 μM cisplatin 0.02, 150 μM + RGCC P = 0.01. G2: 1 μM cisplatin P = 0.02, 10 μM cisplatin 0.01, 0.1 μM cisplatin + RGCC P = 0.02, 10 μM cisplatin + RGCC P = 0.002. Comparing same cisplatin concentration with and without RGCC: S: 0.1 μM cisplatin ± RGCC P = 0.04, G2: 100 μM cisplatin ± RGCC P = 0.009. (b) The effect of 0.1 μM and 1 μM cisplatin with and without 30 μM RGCC in MDAMB231 cell cycle. Compared to control: G1: 0.1 μM cisplatin P = 0.01, 1 μM cisplatin P = 0.0006, 150 μM P = 0.0005, 0.1 μM cisplatin + RGCC P = 0.0009, 1 μM cisplatin + RGCC P = 0.01 10 μM cisplatin + RGCC P = 0.02 S: 1 μM cisplatin P = 0.01, 10 μM cisplatin P = 0.007, 150 μM cisplatin P = 0.04, 10 μM + RGCC P = 0.02. G2: 0.1 μM cisplatin ± RGCC P = 0.008, 1 μM cisplatin P = 0.05, 0.1 μM cisplatin + RGCC P = 0.0008, 10 μM cisplatin + RGCC P = 2 × 10−7, 100 μM cisplatin ± RGCC P = 0.005, 150 μM cisplatin ± RGCC P = 0.05. Comparing same cisplatin concentration with and without RGCC: G2: 100 μM cisplatin ± RGCC P = 0.01|
Click here to view
RGCC small molecule alters gene expression in MCF-7 and MDAMB231
As it can be seen in [Figure 6] both cell lines respond differently to cisplatin. In MCF-7 cells, cisplatin slightly increases ERK1/2 and KRAS but has practically no effect on RAF expression, increases BAX and BCL2 as well as CDKN1A expression. RGCC molecule increases slightly ERK1/2, BAX and BCL2 expression [Figure 6]a. In MBAMB231 cells, cisplatin has no effect on ERK1/2 or KRAS, slightly decreases RAF expression, slightly increases BAX and BAD and increases CDKN1A expression. RGCC molecule decreases ERK1/2, BAX and BCL2 expression [Figure 6]b.
|Figure 6: (a) The effect of cisplatin (0.1 and 1 μM) and RGCC (30 μM) on MCF-7 gene expression. Results show comparison of treated versus untreated cells. (b) The effect of cisplatin (0.1 and 1 μM) and RGCC (30 μM) on MDAMB231 gene expression. Results show comparison of treated versus untreated cells. (c) The effect of cisplatin (0.1 and 1 μM) plus RGCC (30 μM) on MCF-7 gene expression. Results show comparison of cisplatin + RGCC versus cisplatin only treated cells. (d) The effect of cisplatin (0.1 and 1 μM) plus RGCC (30 μM) on MDAMB231 gene expression. Results show comparison of cisplatin +RGCC versus cisplatin only treated cells|
Click here to view
When comparing cisplatin plus RGCC treated cells with cisplatin only cells it can be seen that in MCF-7 cells ERK, RAS and RAF expressions seem to be dependent on cisplatin concentration used, although the differences are very small. However, in this case, the addition of RGCC causes an increase in BAX and BCL2 [Figure 6]c. In MDAMB-231 RGCC addition decreases ERK expression as well as BAX and BCL2. CDKN1A expression is increased when RGCC is added [Figure 6]d.
RGCC small molecule has no effect on total extracellular signal-regulated kinase and phosphoextracellular signal-regulated kinase protein levels in MCF-7 and MDAMB231
Finally, the effect of cisplatin and RGCC small molecule on ERK and phospho-ERK protein levels in both MCF-7 and MDAMB231 cells was determined. There was not statistical difference found in either total ERK or phosphor-ERK in both cell lines (results not shown).
| > Discussion|| |
MCF-7 cells represent luminal A breast tumors that are more differentiated and generally treated successfully with chemotherapy. These cells are also PIK3CA mutated but although there is a mutation in PI3K gene, AKT protein is not activated. MDAMB231 however, represent a claudin-low subtype which is less differentiated and difficult to treat. They are BRAF/KRAS mutated meaning that B-Raf and K-Ras proteins are activated constitutively. It was found that in MBAMD231, ERK levels are higher than those in MCF-7. MDAMB231 is considered to be a good model for TNBC.
The role of ERK pathway is cisplatin is still unclear. Although some researchers state that ERK suppression increases cisplatin resistance, others state that ERK inhibition enhances cancer cell sensitivity to cisplatin. Even ERK activation itself is controversial since it could either prevent or contribute cisplatin's cytotoxic effect.,
We tested an intermediate product in ERK small molecule inhibitor synthesis. Previous experiments have shown that this molecule decreases MCF-7 and MBAMB231 proliferation possibly through phospho-ERK retention in the cytoplasm. Despite the fact that the role of ERK protein in cisplatin treatment is not elucidated yet, we tested the effect of combination therapy of cisplatin plus RGCC small molecule on MCF-7 and MDAMB231 cell lines. Cisplatin was used at concentrations where there was no effect on either cell proliferation or viability.
Combination therapy reduced cisplatin dosage required for a significant decline in both viability and proliferation. Viability reduction is the equivalent of cytotoxic, whereas proliferation reduction denotes cytostatic. For both cell growth and survival, ERK pathway is pivotal. The first demonstration of the role of ERK in cell proliferation took place in 1992, when it was found that ERK1 antisense mRNA or mutant ERK1 that cannot be phosphorylated inhibit cell growth. MEK inhibitor PD184352 was found to abolish cell proliferation due to ERK. Also, ERK1 gene knock-outs in mouse thymocytes had anti-proliferative effects. More recently it was found that Raf kinase inhibitory protein is down-regulated in many cancers.
Cell invasion is indicative of the ability to form metastases. ERK actively participates in cell migration as it was found by the use of respective inhibitors. ERK activation was found to be increased in invasive breast cancer so much so that ERK role as a prognostic factor for disease-free survival was investigated. It has also been shown that ERK-dependent gene expression is required for numerous breast cancer cell lines' migration. It was found that the effect of ERK on cell migration was not through direct signaling but by activating the expression of other proteins such as slug, a potent inducer of cell movement.
The ability of the cancer cell to form clones is perhaps the most clinically relevant assay as it investigates the long-term effects of a substance. It reflects the clinical reality where cancer is not exposed to the drug continuously but at doses. Again, the effect of ERK on proliferation and cell division has been shown in many studies.,,
Next we sought to examine the effect of RGCC small molecule addition on cell cycle. Cisplatin at nontoxic concentrations decreases G1 and increases S and G2 denoting proliferation. This come in agreement with the viability assay, were low cisplatin concentrations increased viability. When RGCC small molecule is added, there is an increase in G1 phase denoting growth arrest. The role of ERK pathway in cell cycle has been well established., ERK activation is responsible for the progression from G0/G1 to the S phase and has been found to be linked to cyclin D activation. Others have found that ERK pathway inhibition induces cell accumulation to the G1 phase. However, as is the case with ERK protein, the different results have also been demonstrated. It was also shown that cell cycle arrest and apoptosis are mediated by ERK activation. The relationship between cell cycle arrest and cytotoxicity is controversial. If anything, cell cycle arrest seems to oppose the effect of cytotoxicity, since cells might survive by entering growth arrest and let DNA repair mechanisms try to fix the DNA damage.
In both cell lines, cisplatin alone in low concentrations caused either no effect or a slight increase in proliferation as evident from both viability and flow cytometry results. On gene level, however, cell proliferation reflects different signaling pathways as shown by PCR. The PI3K mutant (MCF-7) cell line increases ERK expression on cisplatin addition. On the other hand, the PI3K/RAS mutated cell line (MDAMB231) shows no differences in ERK expression upon cisplatin addition. ERK increase could mean both ERK-induced cell death/arrest or proliferation. BAX and BCL2 are increased in both cell lines denoting that the fate of the cell is not strictly apoptosis or proliferation, but those mechanisms coexist simultaneously. In combination treatment, MDAMB231 cells downregulate almost all genes, whereas in MCF-7 cells lower cisplatin down-regulates gene expression as well, whereas higher cisplatin concentration increases gene expression. Changes in gene expression can be either the cause or the results of treatment effect. Whichever is the case, the simultaneous induction of both apoptotic and anti-apoptotic genes denotes that cytotoxicity is a balance between survival and death signals.
Western blot analysis showed that there was no difference in ERK protein levels. Previously we have shown that RGCC small molecule addition at higher concentrations increases phospho-ERK levels in the cytoplasm and decreases nuclear phosphorylated levels. This finding could suggest that our molecule blocks the entry of phospho-ERK in the nucleus where it can activate transcription factors, resulting in cytoplasmic accumulation.
Although ERK pathway is considered to be proliferative, it has been shown that it can also activate cell death. Low-intensity DNA damage induced by ERK causes cell cycle arrest, while extensive damage induces apoptosis. Furthermore, death-associated protein kinase interacts with ERK1/2 and promotes apoptosis. One of the factors affecting the balance between ERK proliferation and ERK cell death is the duration of the signal. However, the signal by itself is not enough. Cell fate determines the biological outcome of ERK activation. Different cells may have different requirements for ERK activation levels and the sensitivity to the duration of ERK activation might be different. Another factor affecting the way ERK controls both cell proliferation and cell death may be found in the localization of ERK. For example, PEA-15 binds to ERK and does not allow it to translocate to nucleus and bind to its transcription factors such as Elk-1, a known proliferative molecule. Therefore, PEA-15 depletion can stimulate ERK-dependent proliferation by allowing ERK nuclear transportation. The differences observed in sensitivity between MCF-7 and MDAMB231 can be attributed to the pathways the cells use for proliferation. MCF7 uses the AKT pathway, whereas MDAMB231 as a KRAS/BRAF mutant line has a constitutively active MAPK as well as AKT pathway. Furthermore, it was found that in KRAS mutated cells, Raf-1 feedback is controlled, whereas Ras is not, indicating that strong feedback mechanisms might exist in MDAMB231 cells that provide robustness to ERK protein levels perturbations.
| > Conclusion|| |
To sum up, RGCC small molecule addition affected MCF-7 and MDAMB231 proliferation and viability by causing cell cycle arrest, their ability to invade and also form clones. Expression of genes implicated in ERK, cell death and cell cycle pathways was also affected. Whether gene perturbation is a result or the cause remains to be investigated in future experiments. Although the decrease in cell proliferation and viability is due to the addition of the ERK small molecule inhibitor, no difference in ERK levels was found. This is indicative of the complex role of ERK in both cell proliferation and cell death, factors inherent to the small molecule itself, such as negative feedback or related to the sensitivity of the method used which cannot detect small protein changes. Although the exact mechanism of action of the combination of cisplatin plus RGCC small molecule inhibitor remains to be investigated, it was found that it decreases dramatically cisplatin dosage needed to cause cytotoxicity. Our findings could potentially lead to a new therapeutic regime for the treatment of cancer.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Torre LA, Islami F, Siegel RL, Ward EM, Jemal A. Global cancer in women: Burden and trends. Cancer Epidemiol Biomarkers Prev 2017;26:444-57.
Park JH, Ahn JH, Kim SB. How shall we treat early triple-negative breast cancer (TNBC): From the current standard to upcoming immuno-molecular strategies. ESMO Open 2018;3:e000357.
Silver DP, Richardson AL, Eklund AC, Wang ZC, Szallasi Z, Li Q, et al
. Efficacy of neoadjuvant Cisplatin in triple-negative breast cancer. J Clin Oncol 2010;28:1145-53.
Agrawal LS, Mayer IA. Platinum agents in the treatment of early-stage triple-negative breast cancer: Is it time to change practice? Clin Adv Hematol Oncol 2014;12:654-8.
Brown A, Kuman S, Tchounwou PB. Cisplatin-based chemotherapy of human cancers. J Cancer Sci Ther 2019;11:97-103.
Dhar S, Kolishetti N, Lippard SJ, Farokhzad OC. Targeted delivery of a cisplatin prodrug for safer and more effective prostate cancer therapy in vivo
. Proc Natl Acad Sci U S A 2011;108:1850-5.
Johnstone TC, Park GY, Lippard SJ. Understanding and improving platinum anticancer drugs-phenanthriplatin. Anticancer Res 2014;34:471-6.
Marques MP, Gianolio D, Cibin G, Tomkinson J, Parker SF, Valero R, et al
. A molecular view of cisplatin's mode of action: Interplay with DNA bases and acquired resistance. Phys Chem Chem Phys 2015;17:5155-71.
Martinho N, Santos TC, Florindo HF, Silva LC. Cisplatin-membrane interactions and their influence on platinum complexes activity and toxicity. Front Physiol 2018;9:1898.
Shirmanova MV, Druzhkova IN, Lukina MM, Dudenkova VV, Ignatova NI, Snopova LB, et al
. Chemotherapy with cisplatin: Insights into intracellular pH and metabolic landscape of cancer cellsin vitro
and in vivo
. Sci Rep 2017;7:8911.
Hoek J, Bloemendal KM, van der Velden LA, van Diessen JN, van Werkhoven E, Klop WM, et al
. Nephrotoxicity as a dose-limiting factor in a high-dose cisplatin-based chemoradiotherapy regimen for head and neck carcinomas. Cancers (Basel) 2016;8. pii: E21.
Mansoori B, Mohammadi A, Davudian S, Shirjang S, Baradaran B. The different mechanisms of cancer drug resistance: A brief review. Adv Pharm Bull 2017;7:339-48.
Yin N, Yi L, Khalid S, Ozbey U, Sabitaliyevich UY, Farooqi AA. TRAIL mediated signaling in breast cancer: Awakening guardian angel to induce apoptosis and overcome drug resistance. Adv Exp Med Biol 2019;1152:243-52.
Florea AM, Büsselberg D. Cisplatin as an anti-tumor drug: Cellular mechanisms of activity, drug resistance and induced side effects. Cancers (Basel) 2011;3:1351-71.
Hatzidaki E, Vlachou I, Elka A, Georgiou E, Papadimitriou M, Iliopoulos A, et al
. The use of serum extracellular vesicles for novel small molecule inhibitor cell delivery. Anticancer Drugs 2019;30:271-80.
Schneider CA, Rasband WS, Eliceiri KW. NIH Image to IMAGEJ: 25 years of image analysis. Nat Methods 2012;9:671-5.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C (T)) method. Methods 2001;25:402-8.
Holliday DL, Speirs V. Choosing the right cell line for breast cancer research. Breast Cancer Res 2011;13:215.
Kabor MH, Suh EJ, Lee C. Comparative phosphoproteome analysis reveals more ERK activation in MDA-MB-231 than in MCF-7. Int J Mass Spectrom 2012;309:1-2.
Yeh PY, Chuang SE, Yeh KH, Song YC, Ea CK, Cheng AL. Increase of the resistance of human cervical carcinoma cells to cisplatin by inhibition of the MEK to ERK signaling pathway partly via enhancement of anticancer drug-induced NF kappa B activation. Biochem Pharmacol 2002;63:1423-30.
Persons DL, Yazlovitskaya EM, Pelling JC. Effect of extracellular signal-regulated kinase on p53 accumulation in response to cisplatin. J Biol Chem 2000;275:35778-85.
Nowak G. Protein kinase C-alpha and ERK1/2 mediate mitochondrial dysfunction, decreases in active Na+transport, and cisplatin-induced apoptosis in renal cells. J Biol Chem 2002;277:43377-88.
Hayakawa J, Ohmichi M, Kurachi H, Ikegami H, Kimura A, Matsuoka T, et al
. Inhibition of extracellular signal-regulated protein kinase or c-Jun N-terminal protein kinase cascade, differentially activated by cisplatin, sensitizes human ovarian cancer cell line. J Biol Chem 1999;274:31648-54.
Hatzidaki E, Parsonidis P, Apostolou P, Daikopoulou V, Papasotiriou I. Novel small molecule decreases cell proliferation, migration, clone formation, and gene expression through ERK inhibition in MCF-7 and MDA-MB-231 breast cancer cell lines. Anticancer Drugs 2019;30:618-27.
Pagès G, Lenormand P, L'Allemain G, Chambard JC, Meloche S, Pouysségur J. Mitogen-activated protein kinases p42mapk and p44mapk are required for fibroblast proliferation. Proc Natl Acad Sci U S A 1993;90:8319-23.
Fischer AM, Katayama CD, Pagès G, Pouysségur J, Hedrick SM. The role of erk1 and erk2 in multiple stages of T cell development. Immunity 2005;23:431-43.
Farooqi AA, Li Y, Sarkar FH. The biological complexity of RKIP signaling in human cancers. Exp Mol Med 2015;47:e185.
Huang C, Jacobson K, Schaller MD. MAP kinases and cell migration. J Cell Sci 2004;117:4619-28.
Mueller H, Flury N, Eppenberger-Castori S, Kueng W, David F, Eppenberger U. Potential prognostic value of mitogen-activated protein kinase activity for disease-free survival of primary breast cancer patients. Int J Cancer 2000;89:384-8.
Chen H, Zhu G, Li Y, Padia RN, Dong Z, Pan ZK, et al
. Extracellular signal-regulated kinase signaling pathway regulates breast cancer cell migration by maintaining slug expression. Cancer Res 2009;69:9228-35.
Siriwardana G, Bradford A, Coy D, Zeitler P. Autocrine/paracrine regulation of breast cancer cell proliferation by growth hormone releasing hormone via Ras, Raf, and mitogen-activated protein kinase. Mol Endocrinol 2006;20:2010-9.
McCubrey JA, Steelman LS, Chappell WH, Abrams SL, Wong EW, Chang F, et al
. Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance. Biochim Biophys Acta 2007;1773:1263-84.
Lev DC, Kim LS, Melnikova V, Ruiz M, Ananthaswamy HN, Price JE. Dual blockade of EGFR and ERK1/2 phosphorylation potentiates growth inhibition of breast cancer cells. Br J Cancer 2004;91:795-802.
Coleman ML, Marshall CJ, Olson MF. RAS and RHO GTPases in G1-phase cell-cycle regulation. Nat Rev Mol Cell Biol 2004;5:355-66.
Massagué J. G1 cell-cycle control and cancer. Nature 2004;432:298-306.
Downward J. Targeting RAS signalling pathways in cancer therapy. Nat Rev Cancer 2003;3:11-22.
Squires MS, Nixon PM, Cook SJ. Cell-cycle arrest by PD184352 requires inhibition of extracellular signal-regulated kinases (ERK) ½ but not ERK5/BMK1. Biochem J 2002;366:673-80.
Tang D, Wu D, Hirao A, Lahti JM, Liu L, Mazza B, et al
. ERK activation mediates cell cycle arrest and apoptosis after DNA damage independently of p53. J Biol Chem 2002;277:12710-7.
Cheng CH, Wang WJ, Kuo JC, Tsai HC, Lin JR, Chang ZF, Chen RH. Bidirectional signals tranduced by DARK-ERK interaction promote the apoptotoc effect of DAR. Rmbo J 2005;24:294-304.
Stork PJ. ERK signaling: Duration, duration, duration. Cell Cycle 2002;1:315-7.
Formstecher E, Ramos JW, Fauquet M, Calderwood DA, Hsieh JC, Canton B, et al
. PEA-15 mediates cytoplasmic sequestration of ERK MAP kinase. Dev Cell 2001;1:239-50.
Fritsche-Guenther R, Witzel F, Sieber A, Herr R, Schmidt N, Braun S, et al
. Strong negative feedback from ERK to RAF confers robustness to MAPK signalling. Mol Syst Biol 2011;7:489.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]