Human colorectal cancer cell lines HT29 (cat. no. JCRB1383) and DLD1 (cat. no. JCRB1382) were purchased from the Japanese Collection of Research Bioresources Cell Bank, gastric cancer cell line KATO III (cat. no. RCB2088) was purchased from the RIKEN BioResource Center, 44As3 was obtained from Dr Kazuyoshi Yanagihara (National Cancer Center Hospital, Kashiwa, Japan), and pancreatic cancer cell line BxPC3 from Dr. Kenoki Ohuchida (Kyusyu University, Fukuoka, Japan). These cells were cultured in RPMI 1640 medium (Nacalai Tesque, Inc.) supplemented with 10% FBS (Nichirei Biosciences, Inc.) and 1% penicillin-streptomycin (Fujifilm Wako Pure Chemical Corporation). The details of the other human cancer cell lines used in the present study are shown in Table I. The murine colon cancer cell line C26 (cat. no. RCB2657) was obtained from the RIKEN Cell Bank and cultured in RPMI1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin. Murine myoblasts C2C12 (cat. no. CRL-1772), obtained from the American Type Culture Collection and were grown in growth medium (GM) consisting of DMEM (Nacarai Tesque, Inc.) supplemented with 10% FBS and 1% penicillin-streptomycin. Myoblast differentiation was induced by replacing GM with differentiation medium (DM) consisting of DMEM supplemented with 2% horse serum (MillporeSigma) and 1% penicillin-streptomycin or conditioned medium (CM) prepared as described in CM preparation. All cell lines were incubated under standard culture conditions in a humidified 5% CO₂ incubator at 37°C. The cell lines were confirmed to be free of Mycoplasma contamination for at least 6 months.

Cancer cells were seeded at a density of 1.5-2.0×10⁶ cells in 100-mm culture dishes (Corning, Inc.) and cultured in growth medium for 2-3 days. When the cancer cells reached 50-60% confluence, they were washed with PBS and 10 ml of fresh DM was added. After 48 h, when the cells reached 80-90% confluence, the culture medium was collected and centrifuged at 1,000 × g for 10 min at room temperature, and stored at −80°C until use. Finally, a CM consisting of 33% cancer cell culture medium and 66% fresh DM was prepared.

C2C12 cells were treated with 5 μM dorsomorphin (cat. no. S7840; Selleck Chemicals) at the time of inducing differentiation with DM or CM. After 1 h of treatment, the medium containing dorsomorphin was removed and fresh DM or CM was added. DMSO was used as a vehicle control at a final concentration of 0.05%.

Transient transfection was performed in HT29 cells using 15 nM control siRNA (ON-TARGETplus Non-targeting siRNA; cat. no. D-001810-01-05; Revvity) or 15 nM BMP4 siRNA (ON-TARGETplus Human BMP4 siRNA SMARTpool; cat. no. L-11221-00-0005; Revvity). The BMP4 siRNA SMARTpool consists of four siRNA duplexes targeting human BMP4, with the following sequences (5' to 3'): GAGCCAUGCUAGUUUGAUA, UAGCAAGAGUGCCGUCAUU, CGACACUUCUGCAGAUGUU, and CAGGAUUAGCCGAUCGUUA. The non-targeting control siRNA, designed not to target any known human gene, has the following sequence (5' to 3'): UGGUUUACAUGUCGACUAA. Lipofectamine^® RNAiMAX (Thermo Fisher Scientific, Inc.) was used as the transfection reagent according to the manufacturer's protocol. After 24 h of transfection at 37°C in a humidified 5% CO₂ incubator, cells were washed with PBS and the medium was changed to DM. The culture medium was collected for CM and ELISA after 48 h of incubation.

C2C12 myoblasts were cultured in DM or CM for 5 days, and the number of viable cells was determined daily using the trypan blue exclusion test as described previously (41). Briefly, the cells were detached using 0.05% trypsin-EDTA, collected after neutralization with growth medium, and resuspended to single-cell suspension for counting. An aliquot (10 μl) of the cell suspension was mixed with 0.4% trypan blue (cat. no. T8154; Merck KGaA) and the number of living cells was determined using a TC20 cell counter (Bio-Rad Laboratories, Inc.). The number of cells was counted every day from day 0 to day 5.

C2C12 myotubes on sterile glass coverslips were washed in PBS and fixed with 4% paraformaldehyde (cat. no 163-20145; FUJIFILM Wako Pure Chemical Corporation) for 15 min at room temperature followed by permeabilization with 0.2% Triton X-100 in PBS for 10 min at room temperature. Samples were blocked with 5% donkey serum (cat. no SIG-D9663; MilliporeSigma) and 1% BSA (cat. no 01-2030-2; MilliporeSigma) in PBS for 60 min at room temperature, and then incubated with primary MYH antibody (1:100; cat. no. sc376157; Santa Cruz Biotechnology, Inc.) overnight at 4°C, followed by incubation with Donkey Anti-Mouse IgG H&L (Alexa Fluor 488) secondary antibody (1:200; cat. no. ab150105; Abcam) for 1 h at room temperature. Nuclei were stained with DAPI (cat. no. ab-104139; Abcam) for 5 min at room temperature. Images were captured using a Zeiss LSM 880 Fast Airyscan Confocal and analyzed using IMARIS (Oxford Instruments). The differentiation index was calculated as the percentage of nuclei expressing MYH cells relative to the total nuclei. The fusion index was calculated as the percentage of nuclei in multinucleated cells with two or more nuclei relative to the total number of nuclei. These indices were determined by randomly analyzing at least 10 images from each sample.

Whole-cell lysates were extracted using lysis buffer (150 mM NaCl; 50 mM Tris-HCl; pH 7.5; 2 mM EDTA; 1% Triton X-100; 1% sodium deoxycholate and 2% sodium dodecyl sulfate) containing protease inhibitors (Roche Diagnostics) and phenylmethylsulfonyl fluoride (Roche Diagnostics). The total protein concentration was determined using Protein Assay Dye Reagent (Bio-Rad Laboratories, Inc.) according to the manufacturer's protocol. The samples were dissolved in NuPage LDS sample buffer (Thermo Fisher Scientific Inc.) and 1 M dithiothreitol, and then heated for 5 min at 95°C. Proteins (20-30 μg) were separated on 5-20% Bis-Tris gels (International Techno Center Co., Ltd.) and transferred to Hybond-ECL membranes (Cytiva). Membranes were blocked with 5% skim milk at room temperature for 60 min and then incubated overnight at 4°C with the following primary antibodies: MYH (1:20,000; cat. no. sc-376157; Santa Cruz Biotechnology, Inc.), MyoD (1:500; cat. no. sc-377460; Santa Cruz Biotechnology, Inc.), myogenin (1:1,000; cat.no. sc-12732; Santa Cruz Biotechnology, Inc.), myomaker (1:1,000; cat. no. NBP2-34175; Novus Biologicals, LLC), myomixer (1:2,000; cat. no. AF4580; R&D systems, Inc.), Pax7 (1:1,000; cat. no. AB_528428; Developmental Studies Hybridoma Bank), Id1 (1:1,000; cat. no. 18475-1-AP, Proteintech Group Inc.), Id3 (1:1,000; cat. no. 10389-1-AP, Proteintech Group, Inc.), p-Smad1/5/8 (1:500; cat. no. 13820, Cell Signaling technology, Inc.), Smad1/5/8 (1:1,000; cat. no. NB600-962, Novus Biologicals, LLC), Smad4 (1:1,000; cat. no. 38454, Cell Signaling Technology, Inc.), BMP4 (1:2,000; cat. no. ab39973; Abcam), MuRF-1 (1:100; cat. no. sc-398608, Santa Cruz Biotechnology, Inc.), LC3B (1:5,000; cat. no. 2775; Cell Signaling Technology, Inc.), GAPDH (1:20,000; cat. no. 60004-1-ig; Proteintech Group Inc.), ACTB (1:1,000; cat. no A1978; Merck KGaA). Membranes were then washed and incubated with the corresponding HRP-conjugated secondary antibodies [goat anti-rabbit IgG (1:3,000; cat. no. 4050-05; SouthernBiotech), goat anti-mouse IgG (1:3,000; cat. no. 1031-05; SouthernBiotech) and donkey anti-sheep IgG (1:1,000; cat. no. HAF016, R&D Systems, Inc.)] for 60 min at room temperature. The signals were detected using the ECL Prime Western Blotting Detection Reagent (Cytiva) and images were acquired using a FUSION-FX7 imaging system (Vilber-Lourmat).

Total RNA was extracted from cells using Isogen II (Nippon Gene Co., Ltd.,) and 1 μg aliquots were reverse-transcribed to cDNA using ReverTra Ace qPCR RT Master Mix (cat. no. FSQ-201; Toyobo Co., Ltd.). qPCR was performed using the CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.) with TB Green Premix Ex Taq™ II Fast qPCR (cat. no. RR830A; Takara Bio, Inc.) according to the manufacturer's protocol. After performing a denaturation step at 95°C for 3 min, PCR amplification was conducted using 50 cycles of 15 sec of denaturation at 95°C, 5 sec, annealing at 60°C and 10 sec of extension at 72°C. Quantitative values were calculated using the 2^−ΔΔCq (42) method and normalized to the expression of ACTB and GAPDH. RT-qPCR primers were designed for either human or mouse genes depending on the experimental system. Primers for MyoD, myogenin, myomaker, myomixer, Id1, Id3, Pax7 and GAPDH were specific for mouse genes and used for C2C12 cells, whereas primers for BMP family genes and IL-6 were designed for human genes. The primers are listed in Table II.

BMP4 concentration in the culture supernatant from cancer cells was determined in triplicate using a Human BMP4 Quantikine ELISA kit (cat. no DBP400; R&D Systems, Inc.) according to the manufacturer's protocol.

The data were analyzed using JMP Pro 14 (SAS Institute, Inc.). To compare two groups, differences in mean values were evaluated using a two-tailed unpaired Student's t-test. For comparisons of three or more groups, ANOVA followed by Dunnett's or Tukey's post hoc tests was performed. P<0.05 was considered to indicate a statistically significant difference and all results were expressed as the means ± SD.

The present study first investigated the morphological changes in C2C12 cells cultured in DM or CM from cancer cells for 5 days. From the beginning of C2C12 differentiation induction in DM or CM (designated as day 0), the medium was changed every 24 h (Fig. 1A). C2C12 myoblasts cultured in DM fused with each other and transformed into myotubes with multiple nuclei on days 3 and 5 (Fig. 1B). By contrast, C2C12 cells cultured with CM from C26, formed fewer myotubes on day 5 (Fig. 1B). Next, the inhibitory effects of CM from 20 human GIC cell lines on myotube formation in C2C12 cells was explored. As shown in Fig. 1C, CM from the colorectal cancer cell lines HT29 and DLD1, the pancreatic cancer cell lines BxPC3 and the gastric cancer cell lines KATOIII and 44As3, inhibited the myogenic differentiation of C2C12 cells. By contrast, CMs from the remaining GIC cell lines exhibited either minimal or weak inhibitory effects on myoblast differentiation (Fig. S1).

To investigate the mechanism underlying the inhibitory effect of CM from GIC on myoblast differentiation, subsequent analysis focused on HT29 and DLD1 cell lines, which exhibited the most pronounced inhibitory effects on C2C12 morphology based on visual assessment in the initial screening (Figs. 1C and S1). The 58As9 cell line, which did not inhibit the differentiation, was used as a negative control.

In C2C12 cells cultured with DM and 58As9 CM, cell growth was markedly suppressed, and mature myotubes with MYH-positive staining appeared on day 5 (Fig. 2A and B). Differentiation and fusion indices were estimated to be >50% (Fig. 2C). By contrast, C2C12 cells cultured with HT29 and DLD1 CMs proliferated with time dependency by day 5 (Fig. 2A). The formation of MYH-positive myotubes (Fig. 2B), and differentiation and fusion indices were significantly suppressed compared with cells cultured in DM and 58As9 CM (Fig. 2C).

Next, the present study evaluated the expression of myogenic genes related to differentiation and cell fusion. In C2C12 cells with controls, the protein expression level of Pax7, which is a key marker for satellite cells and myoblasts, visibly decreased on day 3, whereas the expression of the MRF member MyoD showed a slight reduction. Conversely, the mRNA and protein expression levels of another MRF member, myogenin and its downstream targets, myomaker and myomixer, increased over time (days 1-3) under control conditions (Fig. 2D and E). By contrast, in C2C12 cells cultured with HT29 and DLD1 CM, Pax7 mRNA expression significantly increased, and its protein expression level was preserved on day 3. The expression of MyoD did not show a significant difference when compared with cells cultured in DM and 58As9 CM. However, the expression of myogenin and its downstream targets was remarkably suppressed compared with cells cultured in DM and 58As9 CM (Fig. 2D and E). These results suggest that CMs from HT29 and DLD1 inhibited C2C12 differentiation from myoblasts to myotubes by decreasing the expression of myogenin and its downstream factors. At this point, we hypothesized that some secreted factor from HT29 and DLD1 may exogenously inhibit myoblast differentiation in C2C12 cells by activating the intrinsic signaling pathway.

The present study focused on BMP signaling as a possible mechanism underlying impaired differentiation in C2C12 cells treated with HT29 and DLD1 CMs. First, the mRNA expression levels of BMP family members BMP2, BMP4, BMP6 and BMP7 in control 58As9, HT29 and DLD1 cells were investigated. BMP4 mRNA expression was significantly higher in HT29 and DLD1 when compared with that in 58As9 cells (Fig. 3A). Higher expression of BMP4 protein expression was also observed in cell lysates and culture supernatants from HT29 and DLD1 cells when compared with that in 58As9 cells (Fig. 3B and C). In addition, mRNA expression of the other BMPs was not commonly expressed in HT29 and DLD1 cells (Fig. S2). Next, the present study analyzed whether BMP downstream Smad-Id signaling was activated in C2C12 cells treated with HT29 and DLD1 CM. Expression of p-Smad1/5/8 (pSmad1/5/8), which is the activated form of Smad1/5/8, was visibly higher in C2C12 cells treated with HT29 and DLD1 CMs when compared with the other two controls during differentiation (Fig. 3D). There was no apparent difference in the expression levels of total Smad1/5/8 or Smad4 among all four groups (Fig. 3D). With respect to the Id family, mRNA expression of Id1 and Id3 was observed in C2C12 cells on day 0. Expression of these mRNAs declined in C2C12 cells treated with DM and 58As9 CM on day 1 and 3; however, high expression of Id1 and Id3 was sustained in C2C12 cells treated with HT29 and DLD1 CMs (Fig. 3E). Furthermore, western blotting analysis demonstrated findings consistent with RT-qPCR results for the protein levels of Id1 and Id3 (Fig. 3F). These results indicate that exogenous BMP4, which is secreted by HT29 and DLD1, may inhibit C2C12 differentiation by activating the Smad-Id pathway.

To clarify whether the inhibitory effect of HT29 and DLD1 CMs on C2C12 differentiation is caused by the activation of BMP-Smad signaling, the present study analyzed the reverse effect of an inhibitor of the BMP type I receptor, dorsomorphin. C2C12 cells cultured in HT29 and DLD1 CMs were treated with or without 5 μM dorsomorphin. IFS analysis showed that dorsomorphin markedly increased the number of MYH-positive myotubes on day 5 (Fig. 4A). The differentiation and fusion indices were also increased by this treatment (Fig. 4B). Moreover, the expression levels of myogenin, myomaker and myomixer were markedly restored by dorsomorphin, whereas MyoD expression was slightly decreased (Fig. 4C). The present study further confirmed that the drug treatment decreased the expression of p-Smad1/5/8, Id1 and Id3 in C2C12 cells treated with both HT29 and DLD1 CMs (Fig. 4D-F). These results demonstrated that attenuation of BMP-Smad signaling by dorsomorphin reversed the inhibitory effect of HT29 and DLD1 CM on C2C12 differentiation.

Whether the inhibition of C2C12 differentiation by CM from other human GIC cells (KATOIII, 44As3 and BxPC3) or mouse C26 cells is mediated by the BMP-Smad-Id signaling pathway was next investigated. The morphological changes with or without dorsomorphin in C2C12 cells cultured with CMs from these cells were first analyzed. Dorsomorphin treatment significantly restored MYH-positive myotube formation, along with increased differentiation and fusion indices, in C2C12 cells treated with KATOIII and BxPC3 CMs, as observed in HT29 and DLD1 CMs (Fig. 5A and B). However, this treatment did not affect the inhibitory effects of 44As3 or C26 CMs (Fig. 5A and B). Furthermore, CM from KATOIII and BxPC3, in addition to HT29 and DLD1, significantly increased the expression of Id1 and Id3 mRNAs in C2C12 cells compared to DM or CMs from other remaining cells (Fig. 5C). Finally, BMP4 was highly expressed and secreted not only in HT29 and DLD1 but also in KATOIII and BxPC3 cells, compared with control 58As9 cells. By contrast, 44As3 cells did not express or secrete BMP4 (Fig. 5D and E). These results suggest that the inhibitory effect of KATOIII and BxPC3 CMs on C2C12 differentiation was induced via the BMP4-Smad-Id signaling pathway.

By contrast, C26 cells are known to induce muscle atrophy via UPS and ALS, which are activated by proinflammatory cytokines, including IL-6 and TNFα (8-11). Thus, an experiment to analyze whether CM from 44As3 and C26 caused atrophy in myotubes differentiated from C2C12 cells was conducted (Fig. S3A). Analysis revealed that CM from 44As3 and C26, but not HT29 or DLD1, induced visible atrophy in myotubes, with a significant decrease in myotube diameter (Fig. S3B and C). Higher expression levels of UPS (associated with MuRF-1) and ALS (associated with LC3B-II) were observed in myotubes cultured with 44As3 and C26 CMs than with HT29 and DLD1 CMs (Fig. S3D). In addition, 44As3 cells expressed higher levels of IL-6 mRNA compared with 4 BMP4-expressing GIC (Fig. S3E). These findings suggest that 44As3- and C26-derived IL-6 not only inhibited myogenic differentiation of C2C12 cells but also accelerated UPS- and ALS-dependent atrophy in myotubes.

The present study attempted to confirm whether cancer-derived BMP4 inhibits myogenic differentiation by activating Smad-Id signaling in C2C12 cells. A knockdown analysis was performed in HT29 cells using siBMP4. After siBMP4 transfection, BMP4 expression and secretion were effectively inhibited in siBMP4-HT29 cells, compared with siCtl (Fig. 6A-C). When C2C12 cells were cultured with CM from siBMP4-HT29, myotube formation with multinuclei was remarkably restored relative to siCtl-HT29, and significantly higher indices of differentiation and fusion were observed (Fig. 6D and E). Moreover, the expression levels of myogenin, myomaker and myomixer were apparently increased in C2C12 cells with CM from siBMP4-HT29 (Fig. 6F). Finally, siBMP4-HT29 CM did not elevate the expression of p-Smad1/5/8, Id1 and Id3 in C2C12 cells relative to siCtl-HT29 CM (Fig. 6G and H). Taken together, these results suggest that HT29-derived BMP4 itself inhibited C2C12 differentiation into myotubes by activating the Smad1/5/8-Id1 and -Id3 signaling pathways.