Inhibitory Effects and Mechanism analysis on the Inhibition of Dacarbazine on Melanoma Stem Cells Proliferation in vitro
Melanoma is a growing interest over the past decades and a very difficult obstacle which is needed to be overcome because of its malignant and poor outcome. In this study, we investigated the effects of the dacarbazine (DTIC) on melanoma stem cells regression and subsequent tumor recurrence. Both high and low dosage of DTIC groups was proved to inhibit the growth of melanoma stem cells in vitro through FCM study and MTT assay. In addition, we detected an increase of caspase activities in both high and low DTIC groups, and also stronger expression of pro- apoptotic proteins in melanoma stem cells through western blot analysis. Moreover, a quantitative RT-PCR test of microRNA-200 (miRNA-200) family in cells receiving DTIC showed upregulated miRNA expression, compared to control group. Therefore, the exposure of DTIC could inhibit the growth of melanoma stem cells and thus improve local control and prevent the recurrence or metastases of melanoma, which was possibly undertaken through the miRNA-200 family pathway.
Key words: melanoma; dacarbazine; microRNA; stem cell
Melanoma is a kind of malignant tumor with an increasing incidence over the past thirty years and a rising mortality rate faster than most other types of cancer . It was highly aggressive while melanoma prognosis is depending on the developmental stage when it was detected and interfered . Currently, surgical excision for primary cutaneous melanoma can have excellent results while chemotherapy still takes major part in treating melanoma at late stage .
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Dacarbazine (DTIC), which can be used in a variety of cancers including fibrosarcoma , Hodgkin’s disease , lung cancer , et at., is now considered as the gold standard treatment of advanced melanoma . Signal DTIC clinical application on malignant melanoma was reported to have an efficacy at about 10% . Moreover, it is also reported that DTIC can promote apoptosis and inhibiting growth and invasion of melanoma both in vitro and in vivo [9, 10]. However, advance melanoma was recently found to show resistance on application of DTIC in patients . And the exact underling mechanism of DTIC in melanoma treatment is still not very clear. Thus there is an urgent need to expand their treatment options in order to achieve better therapeutic effect. These circumstances require us to do more for its mechanism of action in-depth discussion.
MicroRNA (miRNA) is a kind of expression of endogenous 18~25 nucleotides in length, small non-coding RNA molecules, which accounted for only 1% of the human genome, is likely to regulate more than 1/3 of the process in human gene expression, modification, transcription and translation [13, 14]. Currently, the miRNA-200 family, including five members such as the miRNA-200a, the miRNA-200b, the miRNA-200c and miRNA-141 has become the researching focus . There is a highly conserved gene clustering phenomenon, the biological characteristics of time and tissue specificity of miRNA-200 family . It has been indicated that miRNA-200 family showed lower expression in liver cancer and renal cell carcinoma [17, 18], but high expression in tumors like bladder cancer and [19, 20]. Currently, miRNA-200 expression in melanoma cell lines didn’t show an inhibition on the intrusion but caused a switching between invasion modes and as a result regulating morphological plasticity and mode of melanoma [21, 22]. MiRNA-200c was reported to result in a higher proportion of cells with amoeboid invasion mode, while in a prominent miRNA-200a expression cells turned to form the elongated intrusion mode . Moreover, Triozzi et al., has recently reported a relationship between with the miRNA levels and the DTIC usage in patients receiving DTIC and interferon . This implies the underling mechanism of DTIC in melanoma treatment.
Taken all the above reports into consideration, it is highly susceptible that DTIC works form different angle through regulating the miRNA levels in patients with melanoma to inhibit the growth of the stem cells. In this paper, we evaluated whether could induce tumor regression and cause subsequent anti-tumor response and whether the levels of miRNA was correlated with DTIC usage in vitro.
Materials and Methods
Stem Cell Obtaining and Culture Conditions
The FEMX-1 cell lines were kindly provided by the third military medical university and stem cells of malignant melanoma were acquired from the FEMX-1 cell lines. The stem cell lines were cultured and maintained in RPMI 1640 medium (Gibco, Life Technologies, UK) with 10% fetal bovine serum (FBS, Gibco) and 2 mM L-glutamine (Gibco) supplemented. The culture condition was set in 37ºC with 5% CO2 in a humidified atmosphere and the melanoma stem cell lines were routinely checked in case of mycoplasma infection.
Grouping and DTIC Exposure
The melanoma stem cells were divided into 3groups. Indicated stem cell lines were seeded in each specified well. Different concentrations DTIC (0, 50 or 100mg/ml) were added and designed as control group, low-DTIC, high-DTIC groups. The samples were harvested and analyzed at 72 h depending on the previous analysis.
Flow Cytometry with PI staining
PI staining was employed to quantify the apoptosis of melanoma cells in each group. Briefly, cells were seeded in 6-well plates (2×105 cells/ml) then stained using PI (kaiji co., ltd. Nanjing, China) fluorescence apoptosis detection kit following the manufacturer’s instruction. Samples were analyzed using a FACSCalibur flow cytometer within 1 h after the staining.
Melanoma stem cells from each group were seeded in a 96-well tissue culture plate at a density of 1×104 cells per well. Cell proliferation of each treatment group was determined by MTT assay performed 72 h. Absorbance was read by using an ELx800 Absorbance Microplate Reader (Bio-Tek instruments, Winooski, VT) at 570 nm. The values of the treated cells were calculated as percentages of the untreated control.
Caspase Activity Assay
The activity of caspase 3/8 in FEMX-1 melanoma stem cells was measured using Caspase-Glo 3/8 assay from Promega. Five thousand cells were seeded in 96-well optical bottom plates with white upper structure (Nunc, Roskilde, Denmark) and treated as described above. The activity was measured after 48 hrs in accordance with the manufacturer’s instructions. Assays were performed in triplicate and repeated at least three times.
Western blot of apoptotic proteins
Western blot assessment was performed to determine the protein expression levels of Bim, Bak, BAX, and Bad. Tissues were obtained as depicted above. The microvessel fractions were isolated from the tumor tissue by centrifugation in 15 ml with 18% (w/v) dextran solution at 10,000g and at 4 °C for 10 min and then were immediately frozen in liquid nitrogen and stored at 280 °C. Proteins from RAECs were homogenized in 10 volumes of lysis buffer (2 mM EDTA, 10 mM EGTA, 0.4% NaF, 20 mM Tris-HCl, 1% NP-40, 1% Triton X-100, protease inhibitors, pH 7.5), the samples were centrifuged (17,000g,4 °C) for 1 hr, and the protein concentration of the soluble material was determined by the Coomassie G250 binding assay. Equal amounts of proteins (10–20lg) were fractionated on 15% SDS-PAGE, followed by transfer to nitrocellulose membranes (Abcam, Cambridge, United Kingdom). The membranes were blocked in blocking buffer (5% nonfat dairy milk dissolved in Tween-Tris-buffered saline [TTBS]) overnight at 4 °C. The blots were then incubated with rabbit polyclonal Bim antibody (diluted 1:800; Santa Cruz Biotechnology), rabbit polyclonal Bak antibody (diluted 1:800; Santa Cruz Biotechnology), rabbit polyclonal BAX antibody (diluted 1:400; Abcam), goat polyclonal Bad antibody (diluted 1:400; Santa Cruz Biotechnology), and mouse polyclonal anti-β-actin antibody (diluted 1:1,000; Santa Cruz Biotechnology) was used as an internal control. Immune complexes were visualized by enhanced chemiluminescence (ECL; Abcam). All the protein bands were scanned by Chemi Imager 5500 V2.03 software, and relative integrated density values (IDVs) were calculated by using a computerized image analysis system (Fluor Chen 2.0) and normalized to the IDV of β-actin.
Quantitative RT-PCR of miRNA-200 family
Total RNA extraction from each group and reverse transcription were performed as previously reported  at 24h, 48h, and 72h. For quantitative RT-PCR, the thermal cycle program used for PCR amplification was as follows: 15-s denaturation step at 95°C and 1-min annealing step at 60°C for 45 cycles. This process was loaded and run in duplicate on a Fast Real-Time PCR System (ABI 7500, Applied Biosystems). The amplified products were separated by 2.5% agarose gel electrophoresis and visualized with ethidium bromide. The resultant gel was imaged by a FluorImager 595 (LI-COR Biosciences, USA). The specificity of each amplified product was identified by the dideoxy sequencing methods using Thermo Sequenase Cycle Sequencing Kit, with a DSQ-1000L sequencer (Shimazu Co., Shiga, Japan).
All data are presented as the Mean±Standard deviation (SD). SPSS 16.0 software (SPSS, Chicago, IL, USA) was employed to determine the statistical significance between samples by using analysis of variance and Dunnett’s t-tests.
Melanoma Stem Cell Growth was inhibited after DTIC Exposure in vitro
PI staining in FCM are used to examine whether DTIC exposure could inhibit the growth of melanoma stem cell and induce apoptosis in vitro. As shown in Figure 1, in control group, the apoptosis rate of melanoma stem cell is very low, respectively (0.3 ± 0.02) %. The apoptosis rate of melanoma stem cell in low-DTIC group and high-DTIC group is significantly increased, to (14.7 ± 0.4) % and (19.5 ± 0.8) % respectively. There is a statistically significant difference when comparing with control group (P<0.05); there was also significant difference of apoptosis rate between low-DTIC group and high-DTIC group (P>0.05).
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Statistical data of the MTT analysis are plotted for melanoma in each group receiving distinctive treatment. The inhibition rate of melanoma stem cell is shown Figure 2. Control group showed only slightly cytotoxic effects; low-DTIC group and high-DTIC groups showed various degrees in growth inhibition. The inhibition rates during the 72h were respectively: (4.2 ± 0.1) %, (13.2 ± 0.5) % and (24.4 ± 1.3) %. The results suggest that: there is a statistically significant difference in control group, low-DTIC group and high-DTIC groups (P<0.05); the inhibitory effect of high-DTIC groups against melanoma stem cell growth was significantly better than low-DTIC group (P<0.05).
DTIC Enhanced the Level of Caspase Dependent Apoptosis in Melanoma stem Cells
Subsequently, caspase initiation and activation effect was detected using Caspase-Glo 3/8 assay from Promega. External caspase cascade in melanoma stem cells caused by caspase-8 cracking (Figure 3) after DTIC treatment when compared with control group (P<0.05). Moreover, high-DTIC group induced stronger caspase-8 processing, and the appearance of cracking product band on behalf of caspase-3 the corresponding effective form of caspase in compare with low-DTIC group but the difference shows no significance (P>0.05).
Effect of DTIC on the Expression on Apoptosis Relevant Proteins of Melanoma Stem Cells
In addition, the evaluation of intrinsic pathway was studied by evaluation of Bid cleavage, differences in expression of pro-apoptotic protein, BimEL BimS, Bak, Bax and Bad were all observed in each group (Figure 4). Control group showed low levels of pro- apoptosis proteins than the other two group (P<0.05). The levels of pro- apoptosis protein were higher in high-DTIC group in compare with low-DTIC group. In conclusion, these results show that the onset of pro- apoptotic protein enhances the activation of the intrinsic pathway of the inhibited growth of melanoma stem cells.
Expression Levels of miRNA-200 Family Associate with DTIC Treatment
Levels of miRNA-200 family were demonstrated to be positively correlated with DTIC treatment (Figure 5). All of the miRNA-200 family tested increased significantly (P<0.05) when compared with control group.
In this study, we tested whether DTIC was effective in inhibiting melanoma stem cells, which could induce primary tumor regression and subsequent antitumor response in melanoma treatment. Our results confirmed both low and high levels of DTIC can suppress melanoma stem cell growth in comparison with control group (p<0.05). And high-DTIC group induced a more significant apoptosis relative to low-DTIC groups (p<0.05). Moreover, high-DTIC group also correlates with upregulated pro- apoptosis proteins of melanoma stem cells and caspase reaction. The miRNA-200 family also showed a positive correlation with the DTIC dosage.
Melanoma is a kind of tumors formed by the melanin cell, originated from neural ectoderm . The challenge of the treatment of melanoma is because of its aggressive early multiple metastases . Lesional resection is a very effective intervention, but this is only useful in the early stage . DTIC, which has long been applied as the treatment for metastatic melanoma, Hodgkin disease and sarcoma more than 30 years, is still a first line treatment for the late phase malignant melanoma . DTIC is commonly used as a single agent, along or combined with other drugs in the treatment of metastatic melanoma [7, 9, 25]. Engesæter et al., has demonstrated that DTIC along with Lexatumumab could cause a synergistic anticancer effects in melanoma in ten different cell lines . However, the detailed mechanism of DTIC in metastatic melanoma is still not fully understood. In our study, DTIC alone also showed a considerable antitumor effect in melanoma stem cells in different degree according to different dosages applied. And the antitumor effect was demonstrated to be accomplished by inducing apoptosis of melanoma stem cells.
MiRNAs, which is a kind of RNA that can’t be translated into protein functional small RNA molecules, are the most important members of the coding RNA regulating the expression of the genes in the transcription level [23, 27]. At present, a large number of studies have shown that the miRNA widely participate in a series of activities of tumor cells and are related to environment change in the occurrence, development and prognosis of tumor [23, 28]. The miRNAs showed a lower expression in certain tumors, such as liver cancer and renal cell carcinoma, while a high expression in some tumors (eg, bladder, cervical cancer) [17-20]. Elson-Schwab et al., has reported a general role of miRNA-200 family in suppressing invasion and metastasis of melanoma cells, and highlighted novel distinguishing characteristics of individual miRNA-200 family members . Moreover, Triozzi et al., also reported an association of miRNA levels with DTIC in patients who have melanoma . In this research, we found that levels of miRNA-200 family have been related to DTIC usage in treatment of melanoma stem cells in a dosage dependent manner. Thus miRNA-200 family may be a potential pathway through which DTIC induced apoptosis of melanoma stem cells takes place.
In conclusion, the exposure of DTIC could inhibit the growth of melanoma stem cells and thus improve local control and prevent the recurrence or metastases of melanoma, which was possibly undertaken through the miRNA-200 family pathway. Figure legends
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