Tumor microenvironment is now widely accepted to influence the fate of cancer cells and plays crucial roles in regulating tumor dormancy and chemoresistance. Standard cell culture system on plastic surfaces neglects the important of cell–extracellular matrix (ECM) interactions thus results in a less reliable approach to analyze cellular activity ex vivo. In this study, A549 lung cancer cells were cultured in soft semi-solid growth substrate (Matrigel) to mimic tumor microenvironment and investigate the role played by ECM proteins and the mechanism of cell–ECM communication. A549 cells cultured in semi-solid Matrigel exhibited a decrease in cell proliferation, cell movement, and invasion rate compared with the corresponding cells cultured on plastic plates. Exposure of A549 cells to ECM proteins confers resistance to conventional chemotherapeutic drugs that target actively dividing cells (Etoposide, Paclitaxel, Vinblastine, Doxorubicin, and 2-Deoxy-D-glucose). Cell cycle distribution analysis indicated that growth of cells in semi-solid Matrigel caused a larger percentage of cells in G0/G1 phase (G0/G1 arrest, dormancy). RT-qPCR analysis showed that A549 cells cultured in semi-solid Matrigel exhibited a marked decrease in the expression levels of genes that are related to tumor progression and invasion (uPA, uPAR, MMP2, MMP7, MMP9, and CXCR4). Alterations of various signaling pathways, such as p-ERK, p-Akt, and p-STAT3, which could account for the observed physiologic responses of the cells were determined. Inhibition of ERK1/2 and Akt with U0126 and LY-294002, respectively, induced G0/G1 arrest and the accompanying drug resistance. These results demonstrate that ECM proteins drive A549 cells into a drug-resistant dormancy state, most likely, through inhibition of the ERK1/2 and PI3K/Akt pathways. Growth of A549 cells in semi-solid Matrigel offers several advantages in understanding the mechanism accounting for tumor dormancy and drug resistance which may lead to therapeutic approaches that can eliminate these ECM-suppressed cells to prevent disease recurrence. Increasing data suggest that tumor microenvironment has a profound impact on cancer progression and therapeutic responses (Fridman, R., 1990, Weaver, V.M., 1996). ECM, a major component of the microenvironment, composed of a complex mixture of macromolecules (e.g., collagen family, laminin, elastin, fibronectin, proteoglycans, and polysaccharides). Cellular attachment to ECM components triggers intracellular signaling pathways which dictate numerous cellular functions, including cell shape, proliferation, differentiation, survival, migration and invasion (Fridman, R., 1990, Lin, C.Q., 1993). Cell–ECM interaction can influence the pattern of tissue-specific gene expression, leading to different biological phenotype. Exposure of mammary epithelial cells to laminin-rich basement membrane has been shown to induce milk protein (beta-casein) expression through integrins (Streuli, C.H., 1991, Muschler, J., 1999). Factors in the tissue microenvironment have been suggested to influence the expression of the ECM-degrading enzymes, thus modify the ability to metastasize of human colon carcinoma cells at two different implantation sites (Nakajima, M., 1990). Current report demonstrates the role of microenvironment in the expression of urokinase-type plasminogen activator (uPA) and hence the metastatic potential of human renal cell carcinoma KG-2 cells (Gohji, K., 1997).
Metastasis, the spread of cancer to distant locations in the body, is a primary cause of cancer-related death. The existence of the disseminated tumor cells at the secondary organ in a dormant state for prolonged periods of time has been reported previously (Luzzi, K.J., 1998, Naumov GN., 2002). These quiescent dormant cells remain undetectable to the screening methodologies and evade conventional therapies targeting actively proliferating cells (Naumov, G.N., 2003). Recent studies demonstrated that the dormant cells can switch to proliferative metastatic growth when the growth conditions are favorable and thus responsible for disease recurrence. Tumor dormancy, resistance to chemotherapy and cancer recurrence remain major problems of cancer treatment. It has been reported that the microenvironment components may determine the fate of cancer cells and play a central role in regulating tumor dormancy and chemoresistance (Fridman, R., 1990, Boyerinas, B., 2013, Ghajar, C.M., 2013). Unraveling the mechanism of ECM-mediated tumor dormancy and its switch to metastatic growth should provide useful targets for the development of therapeutic approaches to eliminate these inactive tumor cells, thus limiting the chance that dormant cells become proliferate years later.
The microenvironment of cells cultured in vitro on traditional plastic substrata is considerably different to that of cells in vivo. Cells require not only nutrients and growth factors, but also an appropriate interaction with the matrix components in order to trigger appropriate intracellular signal transduction. Important signals may be lost when culture cells on plastic surfaces. Recent experiments showed that most cell lines can readily proliferate in a monolayer on plastic plates, but do not develop tumors in vivo (Naumov, G.N., 2002). In addition, standard cell culture on plastic surfaces fails to predict realistic response against drug candidates. However, most studies were performed with cancer cells grown on plastic substrata. Thus the culture condition that recreates the appropriate environment for cells should provide a better model for cellular activity studies. In this study, we have adopted a model that mimics tumor microenvironment and assess how the ECM components regulate cellular behavior, especially tumor dormancy and chemoresistance, and its mechanism of actions.
Matrigel is a biological ECM extracted from mouse EHS (Englebreth-Holm-Swarm) sarcoma, a tumor that rich in basement membrane and ECM proteins. It is composed mainly of laminin (56%), followed by collagen IV (31%), entactin (8%), heparan sulfate proteoglycans (5%), nidogen and small amount of proteases as well as growth factor (Oridate, N., 1996). Matrigel polymerizes at room temperature to form a matrix material resembling the basement membrane found in many tissues and is commonly used as a substrate for cell culture.
In this study, human non-small cell lung cancer A549 cells were cultured in semi-solid Matrigel matrix to allow a close interaction with the ECM proteins, a scenario that better reflect the in vivo microenvironment. Cellular behavior, such as cell migration, invasion, chemosensitivity and growth properties of the cells were assessed. In addition, the signaling pathways mediating the effect of ECM proteins were elucidated to assess the molecular mechanisms implicated in tumor dormancy and drug resistance. Cell culture. Human non-small cell lung cancer (NSCLC) cell line A549 was obtained from the American Type Culture Collection (ATCC, RockVille, MD, USA). The cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum and 1% Antibiotic-Antimycotic. For semi-solid Matrigel cultures, 350 μl of complete medium containing 3×105 cells was gently mixed with 150 μl of Matrigel (Corning, Bedford, MA, USA) (30% v/v), plated into 24-well plate and incubated at 37°C for 3 days. This diluted Matrigel polymerizes at 37°C to form soft semi-solid gel that restricts the movement of the cells, yet is soft enough to allow cells to spread as a monolayer beneath the gel. Cells were extracted from the semi-solid culture environment for further analyses by tapping the plate, aspirate the collapsed gel, wash with PBS, followed by conventional trypsinization. In all experiments, the cells were maintained at 37ºC in a humidified atmosphere containing 5% CO2.
Cell proliferation assay. Cell proliferation was determined by Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, San Diego, CA, USA) to measure the DNA quantification according to the guidelines of the manufacturer. Cells were seeded into 96-well culture plates at a density of 1×104 cells per well in 100 μl of culture medium. Cell proliferation was assessed on day 0, 2, 4, 7 and 9 post plating. Fluorescence-based dsDNA measurement was performed using an excitation wavelength at 485 nm and an emission wavelength at 520 nm by a SpectraMax fluorescence microplate reader (Molecular Devices, Sunnyvale, CA, USA).
Cell motility (wound-healing) assay. Cell migration was investigated by wound-healing assay. Wounds were generated by scratching confluent cultures of cells on plastic plates or cells in semi-solid Matrigel with a sterile 20 µl pipette tip. These wounds were then washed twice with PBS to remove cell debris, covered with complete medium or 30% v/v Matrigel and incubated for further 24 h. Wounds were marked and digitally photographed in the same field at 0 and 24 h after scratching. Wound area was measured with Image J software and the extent of healing was calculated as follows: (wound area at 0 h – wound area at 24 h )/wound area at 0 h ×100.
In vitro invasion assay. The invasiveness of cancer cells was evaluated using Matrigel-coated transwell chambers (8 µm pore size). The upper chamber was seeded with cell suspension (1×105 cells in 200 µl) and 500 µl of conditioned medium prepared from human lung fibroblast (MRC-5) cells was added to the lower chamber. After incubation for 24 h at 37°C, the uninvaded cells on upper surface of the insert membrane were removed with a cotton swab. Cells that invaded to the lower surface of the membrane were fixed with 25% methanol, stained with crystal violet, and acid extracted with 0.1N HCl in methanol. The absorbance at 550 nm was measured.
Cell viability assay. The chemosensitivity of the cells was determined by MTT assay for assessing metabolically active cells as previously described (Alley, M., 1988). Briefly, Cell suspension was seeded into 96-well culture plates at a density of 1×104 cells/100 µl/well, followed by the addition of 100 µl of vehicle (cell culture medium) or various concentrations of cytotoxic agents and incubated for 48 h. Cell culture medium containing MTT (Sigma, St. Louis, MO, USA) was added to each well and incubated for 2 h. The number of viable cells was assessed by determining the A550 nm and subtracted with the A650 nm (reference wavelength). The IC50 is defined as the concentration of a drug that gives 50% decrease in the resulting subtracted absorbance. Assays were performed in quadruplicate wells, and data were expressed as percent viability compared with control.
Cell cycle assay. Cell cycle distribution was investigated using Muse™ Cell Analyzer (Merck Millipore, Billerica, MA, USA). Muse™ Cell Cycle Assay Kit was used according to the manufacturer’s recommendations. Briefly, cells from each condition were washed with PBS and fixed in ice-cold 70% ethanol overnight at −20°C prior to staining. Cells were then washed with PBS, stained with 200 μl of Muse™ Cell Cycle reagent, incubated at room temperature in the dark for 30 min, and processed for cell cycle analysis. Results are expressed as a percentage of cells in each phase of the cell cycle (G0/G1, S, and G2/M) based on differential DNA content. Triplicate independent experiments were performed.
Quantitative RT-PCR. Gene expression level was analyzed by real-time quantitative RT-PCR. Cells were harvested and total RNA isolation was performed using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Approximately 2 μg of total RNA was reverse transcribed to cDNA with the SuperScript® III Reverse Transcriptase (Invitrogen). The RT-qPCR was performed using KAPA SYBR® FAST qPCR Kits (Kapa Biosystems, Wobum, MA, USA) and a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with the following steps: 95°C for 10 min, 95°C for 15 sec, 60°C for 30 sec, and 72°C for 30 sec for a total of 40 cycles. All primers used to amplify genes are listed in Table 1. The level of gene expression relative to an internal control, RPS13 (ribosomal protein S13), was determined using the comparative Ct method (Livak, KJ., 2001)
Immunoblot analysis. Expression level of the signaling proteins was investigated by immunoblot analysis. The cells were harvested by trypsinization and lysed in cell signaling lysis buffer (Merck Millipore) containing protease inhibitors and phosphatase inhibitors (1 mM Na3VO4, 10 mM NaF, and 20 mM β-glycerophosphate). Cell extracts were resolved by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon-P Transfer Membrane (Millipore, Bedford, MA, USA). After blocking with 3% bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) for 1 h at room temperature, the membranes were exposed to indicated primary antibodies for overnight at 4°C. Following incubation with the proper horseradish peroxidase-linked secondary antibody, the protein bands were visualized using ECL reagents (GE Healthcare, Little Chalfont, UK).
Statistical analysis. All experiments were performed in triplicate, and the data was reported as means ± standard deviation. Statistical analysis was carried out using the Student’s T-test. Values of p < 0.05 were considered to be statistically significant.
Results
Morphology of A549 cells grown on plastic plates versus in semi-solid Matrigel. When A549 cells plated on plastic dishes or in semi-solid Matrigel were examined by inverted phase-contrast microscopy, the cells grown in semi-solid Matrigel underwent morphological change within 24 h after plating (Fig. 1) from a typical shape to branched shape cells. This morphological change could be reversed by replating cells grown in semi-solid Matrigel back to plastic plates (data not shown).
Growth of A549 cells grown on plastic plates versus in semi-solid Matrigel. Cell growth characteristic of A549 cells cultured on plastic plates or in semi-solid Matrigel was examined on day 0, 2, 4, 7 and 9 by DNA quantification using a PicoGreen DNA assay. The data showed that the growth dynamics were different between two cell populations: proliferative and quiescent growth characteristics (Fig. 2). A549 cells grown in semi-solid Matrigel showed a little increase in cell number during 9 days of culture, while the numbers of the cells grown on plastic plates were increased up to 4-fold on day 4.
Cell–ECM regulation of migration. Cell–ECM interaction is a critical factor in cell migration and invasion, which are key determinants of cancer progression and metastasis. To investigate the role of ECM proteins in regulation of cell motility, wound-healing assay was performed. Cell migration was expressed as a percentage of wound closure (Fig. 3). The results demonstrated that at 24 h, about 50% of the gap had closed in A549 cells grown on plastic plates and covered with culture medium, while only 30% of the initial gap had closed in cells grown on plastic plates and covered with semi-solid Matrigel. For A549 cells grown in semi-solid Matrigel, only 25% and 10% of the gap had closed when covered with culture medium and semi-solid Matrigel, respectively. These results suggest that A549 cells cultured on plastic plates were more motile than cells cultured in semi-solid Matrigel in both conditions and that the motility was decreased in the presence of Matrigel.
Cell–ECM regulation of invasion. Transwell invasion assays were performed to assess the effect of ECM proteins on cell invasiveness. A549 cells cultured on plastic plates and in semi-solid Matrigel were investigated and the results were clearly shown a significant decrease in invasion rate of cells cultured in semi-solid Matrigel both in the photography and quantitative analysis (Fig. 4). Invading cells were 41% lower in cells cultured in semi-solid Matrigel, as compared with the cells cultured on plastic plates (p<0.001). These observations suggested that ECM proteins are crucial in regulating A549 cell invasion.
Effect of ECM proteins on chemosensitivity of A549 cells. Drug sensitivity of A549 cells cultured on plastic plates and in semi-solid Matrigel was explored. Different anticancer drugs commonly used to treat a variety of cancers were used in this study. Cells were treated with various concentrations of the drugs for 48 h, followed by MTT assay to determine the survival rate. The drug concentration which caused a 50% decrease in cell number (IC50) was calculated. The results demonstrated that growth of cells in semi-solid Matrigel caused a remarkable increase in IC50 to anticancer drugs that target actively dividing cells (Etoposide (10.6 fold), Paclitaxel (5.7 fold), Vinblastine (4.4 fold), Doxorubicin (4.1 fold), and 2-Deoxy-D-glucose (2-DG, 3.9 fold)), as compared with the cells cultured on plastic plates (Table 2), indicating a higher degree of chemoresistance. However, the response of both cell populations was very similar to cytotoxic agents that are not related to cell proliferation rate, Curcumin (1.5 fold) and Bay 11-7085 (0.9 fold). These results indicated the influence of ECM proteins on chemoresistance of A549 cells.
ECM proteins induced G0/G1 cell cycle arrest in A549 cells. To assess the possible mechanisms underlying ECM proteins-mediated cell growth inhibition, cell cycle distribution of A549 cells cultured on plastic plates and in semi-solid Matrigel was investigated. The results demonstrated that cells cultured in semi-solid Matrigel showed higher G0/G1 population (80.1±2.2%) (Fig. 5B) compared with 60.1±1.5% in the cells cultured on plastic plates (Fig. 5A). Consistently, Matrigel exposure caused a decrease in the proportion of cells in S phase and G2/M phase of the cell cycle (6.9±0.8% and 12.7±1.7%) of cells cultured in semi-solid Matrigel as compared with cells cultured on plastic plates (17.5±0.4% and 22.1±1.0%). These data suggested that Matrigel induces cell cycle arrest (cellular dormancy) at the G0/G1 phase, resulting in growth inhibition of A549 cells.
Cell–ECM regulation of gene expression. The change in gene expression of cells upon contact with ECM proteins has been evidenced (Streuli CH., 1991). Therefore, we investigated the consequence of cell–ECM interactions on changes in the expression patterns of genes involved in invasion (MMP2, MMP7, MMP9, MMP11, MMP13, HGF, and CXCR4) and drug resistance (MDR-1 and MRP3). In addition, uPA and uPAR (urokinase plasminogen activator receptor), genes that are related to tumor progression and metastasis were also analyzed. Quantitative real-time RT-PCR was performed to determine the level of gene expression. The relative expression of genes normalized to RPS13 was calculated. Using a threshold value of 3-fold expression change, the results showed that A549 cells cultured in semi-solid Matrigel decreased expression levels of uPA, uPAR, and several genes that linked to invasion (MMP2, MMP7, MMP9, and CXCR4), as compared with the cells cultured on plastic plates. However, the expression levels of drug resistance genes, MDR-1 and MRP3, did not have much difference (Fig. 6).
Cell–ECM regulation of protein expression. To elucidate the signal mediated by cell–ECM communication, the changes of signaling molecules associated with proliferation, metastasis, chemoresistance and apoptosis were assessed. Western blot analysis of proteins extracted from A549 cells cultured on plastic plates and in semi-solid Matrigel indicated that growth of cells in semi-solid Matrigel reduced the expression of p-ERK, p-Akt, and p-STAT3, a positive regulator of cell growth, proliferation, and survival. Bcl-xL, the anti-apoptotic protein, was up-regulated in cells cultured in semi-solid Matrigel. Consistently, a decreased level of the anti-apoptotic protein, Bax, was detected. The level of p-p65 (NF-κB) protein was increased in cells cultured in semi-solid Matrigel compared to cells cultured on plastic plates. In contrast, the level of its inhibitor, IκB, was decreased. Furthermore, the up-regulation of p-p38 and p21 was found in cells cultured in semi-solid Matrigel, while the expression of p-FAK, cyclin D1, uPAR, and MYLK was lower than that in cells cultured on plastic plates (Fig. 7).
Inhibition of MAPK and Akt pathways induced G0/G1 cell cycle arrest and protect against Etoposide-induced cell death. Selective inhibitors of MEK1/2 (U0126), PI3K (LY-294002), and JAK2 (AG-490) were used to investigate whether these signaling pathways are associated with Matrigel mediated dormancy and chemoresistance. The effects of U0126, LY-294002, and AG-490 on cell viability were first evaluated. A549 cells were treated with various concentrations of U0126, LY-294002, and AG-490 then MTT assay was performed to determine cytotoxicity following treatment. The concentration of the inhibitors that gives cell viability higher than 80% was used. Pretreatment with 30 μM of U0126, 20 μM of LY-294002, and 30 μM of AG-490 for 24 hr did not significantly affect A549 cell viability (data not shown). Western blot analysis revealed a decreased phosphorylation of ERK1/2, Akt, and STAT3 following treatment with U0126, LY-294002, and AG-490, respectively for 24 h (data not shown). Cell cycle analysis showed that blockade of ERK1/2 and Akt with U0126 and LY-294002 resulted in G0/G1 phase cell cycle arrest similar to cells cultured in semi-solid Matrigel (MG). Treatment with AG-490 had no effect on cell cycle progression of A549 cells (Fig. 5C-5E). Cytotoxicity assay was performed using Etoposide and Bay 11-7085 as a representative of drugs that are related and not related to cell proliferation rate, respectively. The inhibitors were preincubated 24 hr before and during the cytotoxic assay. After comparing the effect of the corresponding inhibitor on chemosensitivity, U0126 and LY-294002 were found to increase Etoposide resistance similar to cells cultured in semi-solid Matrigel (Table 2) (the IC50 value of cells pre-treated with U0126 and LY-294002 were 299 ± 23.3 μM and 243.3 ± 16.5 μM, respectively). However, the chemoresistance to Etoposide was unaffected by STAT3 inhibition with AG-490 (the IC50 value of cells pre-treated with AG-490 were 30.8 ± 1.2 μM). For Bay 11-7085, treatment with U0126, LY-294002, and AG-490 showed no effect on chemosensitivity (the IC50 value of cells pre-treated with U0126, LY-294002, and AG-490 were 19.0 ± 0.5 μM, 19.4 ± 0.1 μM, and 17.0 ± 1.1 μM, respectively) (Fig. 8). These findings are in agreement with cell cycle distribution that inhibition of ERK1/2 and Akt with U0126 and LY-294002 caused cell dormancy (Fig. 5C-5D) which lead to enhanced chemoresistance for the drugs that target actively proliferating cells. Taken together, these data suggest that ECM proteins-mediated A549 cells growth inhibition and protection from chemotherapy-induced apoptosis was predominantly through MAPK and Akt pathways.
Discussion
Metastasis is the prime cause of death from cancer. Many cancer patients who have no clinical symptoms after primary tumor removal suffer from disease relapse years or decades later (Meltzer, A., 1990, Karrison, T.G., 1999). A likely explanation of cancer recurrence after a long latent period is that the metastatic cells remain dormant in the body, resistant to current therapies and begin reactivation in a permissive microenvironment (Aguirre-Ghiso, J.A., 2007). Various factors have been suggested as possible contributors to cellular dormancy, including complex interactions between metastatic cells and the microenvironment (Yu, W., 1997). Interactions between the metastatic cancer cells and the new organ microenvironment have been shown to influence proliferative properties of cancer cells at the metastatic site (Radinsky, R., 1995, Fidler, I.J., 2001). Recent experiments showed that cell-ECM interactions inhibit proliferation of metastatic melanoma cells (Henriet, P., 2000, Roth, J.M., 2005).
Cells cultured on basement membrane extract have been shown to exhibit morphology and growth characteristics that correlate with the behavior of the cells at secondary sites in vivo (Li, X., 1994, Barkan, D., 2008). Cellular morphology of A549 cells was considerably different between cells cultured on plastic plates and cultured in semi-solid Matrigel in this study. Our results revealed that A549 cells cultured in semi-solid Matrigel exhibited some characteristics similar to that found in dormant cancer cells, as shown by a decrease in cell proliferation, cell motility, and invasion and increase in chemoresistance. Cell cycle progression analysis suggest that growth of A549 cells in semi-solid Matrigel induces G0/G1 cell cycle arrest. Results from our study suggest that semi-solid Matrigel culturing system represents a useful model to study cellular dormancy which may lead to new therapies that can eliminate these dormant cancer cells.
MAPK/ERK, PI3K/Akt, and STAT3 have been implicated in cellular growth, proliferation, and survival. Western blot study revealed the marked reduction in p-ERK, p-Akt, and p-STAT3 expression, along with the decreased proliferation rate of A549 cells cultured in semi-solid Matrigel compared with that in cells cultured on plastic plates. The reduction in ERK1/2 activation has been linked to tumor dormancy in vivo (Aguirre Ghiso et al., 1999, 2001). Recent study demonstrated that the activation of p38 stress-activated kinase induces cell cycle arrest via inhibition of ERK1/2 signaling and uPAR expression (Aguirre Ghiso et al., 2001), consistent with the enhanced phosphorylation of p38 MAPK investigated by western blot analysis and the reduction of uPAR at both mRNA and protein levels. uPA and its receptor, uPAR, have been associated with metastasis, tumor progression, and reduced overall survival of the patients. Current evidence demonstrates that down-regulation of uPAR led to a reduction in FAK phosphorylation and deactivation of ERK1/2, resulting in dormancy in vivo (Aguirre Ghiso et al., 1999, 2002, Yu, W., 1997). The decrease in FAK phosphorylation and uPA mRNA level in this study further support this notion. In support of a role for FAK and myosin light-chain kinase (MYLK, MLCK) in cell migration, the low-motility A549 cells cultured in semi-solid Matrigel showed a decrease in both MYLK and p-FAK protein levels. A recent report has shown that MYLK also has a functional role in proliferative growth, inhibition of MYLK prevents the transition from dormancy to proliferative state (Barkan, D., 2008). The expression level of genes that linked to cancer cell invasion (MMP2, MMP7, MMP9, and CXCR4) also decreased, associated with the decrease in invasion rate of cells cultured in semi-solid Matrigel, compared with the cells cultured on plastic plates.
Cyclin D1 facilitate the cell cycle by sequestering CDK inhibitors, p21, allowing S phase initiation and progression. In agreement with the decrease in proliferative activity of A549 cells cultured in semi-solid Matrigel, cyclin D1 protein level was decreased together with the increased p21 level, thus prevents cells from reentering the cell division cycle. In a recent report, the decrease in p-STAT3 and p-ERK expression leads to down-regulation of the downstream target molecules, including cyclin D1 (Modi, P.K., 2012). p21 was shown to be negatively regulated by Akt (Rössig, L., 2001), then the inactivation of Akt further promote the inhibitory effect of p21 on cell cycle progression. The changes in these signaling molecules are likely attributed to a dormancy of A549 cells cultured in semi-solid Matrigel.
The accumulating evidence for ECM-mediated cytoprotective has been reported (Rintoul, RC., 2002, Hodkinson, P.S., 2007). The response of metastases in different organ microenvironments to chemotherapy was markedly different by the interaction of metastatic cells with local environment factors (Fidler, I.J., 1994). Soluble cytokines secreted by the tumor microenvironment may activate the signal transduction pathways that influence the response to cytotoxic drugs of tumor cells (Dalton, W.S., 1999). ECM-mediated chemoresistance was supported by related observations that adhesion of small cell lung cancer cells to laminin enhanced resistance to several cytotoxic drugs (Fridman, R., 1990). Etoposide treatment of SCLC cells grown on fibronectin significantly reduced caspase-3 activation and apoptosis (Buttery, R.C., 2004). The ECM-mediated chemoresistance in this study is likely due to the decrease in cell proliferation, since A549 cells cultured in semi-solid Matrigel showed remarkably resistance to chemotherapy drugs that are related to cell proliferation rate, while similar results were obtained for drugs that are not related to cell proliferation rate. Targeting these ECM-suppressed cells by compounds that are more potent against cells cultured in semi-solid Matrigel would represent candidates for cancer treatment.
The up-regulation of Bcl-xL and the down-regulation of Bax may also be involved, at least in part, in evasion of apoptosis of A549 cells cultured in semi-solid Matrigel. In addition, p-p65 has been reported to confer resistance to chemotherapy (Godwin, P., 2013). The increased level of p-p65, together with the decreased level of IκB could also responsible for the acquired chemoresistance of A549 cells cultured in semi-solid Matrigel. Overexpression of drug efflux pumps was associated with chemoresistance. However, the mRNA expression levels of MDR-1 (encodes P-glycoprotein) and MRP3 (encodes multi-drug resistance protein) was unaffected by ECM proteins in this study. Cell cycle progression analysis and cytotoxicity assay indicated that blockade of ERK1/2 and Akt induced G0/G1 phase cell cycle arrest and the accompanying chemoresistance similar to cells cultured in semi-solid Matrigel. These findings are in agreement with previous studies that inhibition of ERK1/2 and Akt induce dormancy and correlated with acquired resistance to chemotherapy drugs (Endo, H., 2014, Li, Q., 2009).
Taken together, our results suggest that MAPK/ERK and PI3K/Akt might be the possible mechanism underlying Matrigel mediated dormancy and chemoresistance of A549 cells. The semi-solid Matrigel culture environment used in this study can induce a dormancy-like phenotype which may mimic in vivo dormancy and response against chemotherapy. This culture system could provide a means for screening and develop new therapeutic approaches to improve the response to chemotherapeutic agents and eliminate dormant micrometastatic cells. Figure 1 Morphological comparison of A549 cells cultured on plastic plates and in semi-solid Matrigel. A549 cells were seeded at 5 × 104 cells/ml in 24 well plates either on plastic (MD) or in semi-solid Matrigel (MG) and examined every 24 h for 3 days by inverted phase-contrast microscopy with digital camera. A549 cells cultured on plastic plates maintain the typical cobblestone shape, while the cells cultured in semi-solid Matrigel adopt a branched morphology. Images were captured at a magnification of ×200, scale bar: 100 µm.
Figure 2 Effects of ECM proteins on cell proliferation. A549 cells were cultured on plastic plates or in semi-solid Matrigel. Cell numbers were quantitatively measured on day 0, 2, 4, 7 and 9 using a PicoGreen DNA assay. The growth of A549 cells cultured in semi-solid Matrigel (MG) was greatly attenuated compared with cells cultured on plastic plates (MD). Data are presented as the mean ± SD. The data are representative of three independent experiments with similar results.
Figure 3 Effects of ECM proteins on motility of A549 cells. Confluent monolayers of A549 cells cultured on plastic plates (MD) or in semi-solid Matrigel (MG) were scratched using a pipette tip and then covered with culture medium (md) or semi-solid Matrigel (mg). Photographs were taken at 0 and 24 h after scratching. Quantification of the wound closure during 24 hr of cell migration showed that matrigel suppresses the motility of A549 cells which resulted in reduced gap closure. Data are presented as the mean ± SD of three independent experiments. ** p<0.01, *** p<0.001. Images were captured at a magnification of ×100, scale bar: 100 µm.
Figure 4 Effects of ECM proteins on invasion of A549 cells. The cells were cultured on plastic plates or in semi-solid Matrigel before subjected to Transwell invasion assays. Cells were allowed to invade for 24 h. The percentage of invasive A549 cells was significantly decreased in cells cultured in semi-solid Matrigel (MG), compared with cells cultured on plastic plates (MD). Data are shown as the mean ± SD of three independent experiments. *** refers to a significant difference (p<0.001). Images were captured at a magnification of ×200, scale bar: 100 µm.
Figure 5 Analysis of cell cycle distribution of A549 cells. Fractions of G0/G1, S, and G2/M cell population were determined using Muse™ Cell Analyzer. (A) Cell cycle profiles of A549 cells cultured on plastic plates (MD). (B) Cell cycle profiles of A549 cells cultured in semi-solid Matrigel (MG). (C) Cell cycle profiles of A549 cells treated with 30 μM of U0126 for 24 h. (D) Cell cycle profiles of A549 cells treated with 20 μM of LY-294002 for 24 h. (E) Cell cycle profiles of A549 cells treated with 30 μM of AG-490 for 24 h. One representative of three independent experiments with similar results is shown.
Figure 6 Quantitative real-time PCR analysis of genes in A549 cells cultured in semi-solid Matrigel compared with cell cultured on plastic plates. Relative quantification was calculated from the Ct values. Data are expressed as fold up-regulation of genes expressed by A549 cells cultured in semi-solid Matrigel over those expressed by cells cultured on plastic plates. The expression of UPA, UPAR, and several genes that linked to invasion (MMP2, MMP7, MMP9, and CXCR4) was significantly decreased in A549 cells cultured in semi-solid Matrigel. The relative level of gene expression was calculated using the comparative Ct method, normalized to the expression of the reference gene RPS13. All data are mean ± SD of three independent experiments. * indicates significant difference (at least 3-fold difference).
Figure 7 Immunoblot analysis of total extracts from A549 cells cultured in semi-solid Matrigel (MG) compared with cell cultured on plastic plates (MD). 40 μg of whole cell extracts were subjected to immunoblotting with the indicated monoclonal antibodies. Data shown are representative immunoblots from at least three separate experiments. Expression of GAPDH was used as loading control.
Figure 8 Effects of the selective inhibitors (U0126, LY-294002, and AG-490) on chemosensitivity to Etoposide and Bay 11-7085. Cells were preincubated with U0126 (30 μM), LY-294002 (20 μM), and AG-490 (30 μM) 24 hr before and during the cytotoxic assay. Cells were treated with various concentrations of the drugs for 48 hr and cytotoxicity was determined by using MTT assay. Data are presented as mean ± SD of three independent experiments.