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. 2026 Aug 28;481(9):5087–5099. doi: 10.1007/s11010-026-05714-8

Lactic acid exposure alters lysophosphatidic acid receptor–mediated regulation of growth and motility in pancreatic cancer cells

Mao Yamamoto 1,#, Miwa Takai 1,#, Nanami Shimomura 1, Eiki Isa 1, Narumi Yashiro 1, Moemi Tamura 1, Shion Nagano 1, Yuka Kusumoto 1, Toshifumi Tsujiuchi 1,✉
PMCID: PMC13638741  PMID: 42663874

Abstract

Lysophosphatidic acid (LPA) receptor signaling contributes to the regulation of cancer cell functions. Lactic acid serves not only as an important energy source for cancer cells but also activates various signaling pathways that influence cancer cell behavior. To investigate the roles of LPA receptors in the malignant behavior of pancreatic cancer PANC-1 cells, PANC-LA1 and PANC-LA5 cells were generated by culturing PANC-1 cells with 1 or 5 mM lactic acid, respectively, for approximately 3 months. LPAR1, LPAR3, and LPAR5 expression levels expression levels were elevated, whereas LPAR2 expression was reduced in PANC-LA1 and PANC-LA5 cells compared with PANC-1 cells. PANC-1 cell growth was inhibited by LPA. Conversely, PANC-LA1 cell growth remained unchanged in response to LPA, and LPA increased PANC-LA5 cell growth. The motility of PANC-LA1 and PAN-LA5 cells was markedly elevated compared with that of PANC-1 cells. LPA further enhanced PANC-LA1 and PAN-LA5 cell motility but did not affect PANC-1 cell motility. In the presence of LPA, AM966 inhibited, whereas TC LPA5 4 stimulated PANC-LA5 cell growth and PANC-LA1 cell motility. Although GRI-977,143 increased PANC-LA5 cell growth, it reduced PANC-LA1 cell motility. (2S)-OMPT increased PANC-LA5 cell growth and PANC-LA1 cell motility. When cells were cultured in serum-free media, the viability of PANC-LA1 and PANC-LA5 cells was higher compared with that of PANC-1 cells. PANC-LA5 cell growth and PANC-LA1 cell motility were inhibited by AZD3965 and galloflavin. These findings indicate that LPA receptor signaling is involved in the regulation of PANC-1 cell behavior induced by long-term lactic acid exposure, with LPA1 and LPA3 acting as major positive regulators of proliferation and motility following long-term lactic acid exposure, whereas LPA2 and LPA5 exert distinct regulatory effects.

Keywords: Cellular functions, Lysophosphatidic acid, LPA receptors, Lactic acid, Pancreatic cancer cells

Introduction

Lactic acid is a waste cellular product generated through glycolytic and oxidation metabolism. In cancer cells, glucose functions as a major metabolic substrate supporting ATP generation, redox homeostasis, and biosynthetic processes. Glucose metabolism can proceed through cytoplasmic glycolysis, producing lactate, or through mitochondrial oxidative phosphorylation, generating carbon dioxide and water [1, 2]. Notably, many cancer cells preferentially rely on glycolysis even under oxygenated conditions, a metabolic phenotype known as the Warburg effect, which results in increased lactate production [1, 3]. Furthermore, metabolic heterogeneity exists within tumors, where subsets of cells predominantly utilize glycolysis while others depend more heavily on oxidative phosphorylation. Metabolic interactions between tumor cells and surrounding stromal cells also facilitate the exchange of metabolites that sustain tumor cell activity [1, 4]. Recently, it has been recognized that lactic acid serves not only as an essential energy source supporting tumor cell viability but also as a mediator that activates several cellular signaling pathways in the tumor microenvironment (TME). Accumulating evidence indicates that lactate acts as a signaling molecule that modulates cellular communication, metabolic adaptation, and malignant progression within the TME [5, 6].

Lysophosphatidic acid (LPA) receptors (LPA1 to LPA6) are activated through the binding of the lipid mediator LPA, which consists of a glycerol backbone linked to a fatty acid and a phosphate group [7–9]. So far, several studies have shown that LPA receptor-mediated signaling enhances malignant cancer cell properties, including cell growth, motility, invasion, and resistance to chemotherapy [10]. In the TME of solid tumors, abnormal vascular structures restrict oxygen supply from blood vessels, thereby generating hypoxic and nutrient-deprived conditions for cancer cells [11, 12]. Our previous work has demonstrated that LPA receptor-mediated signaling regulates cancer cell functions under hypoxic and nutrient-deprived conditions [13–15]. These findings suggest that LPA receptor signaling plays important roles in the adaptive responses of cancer cells to the metabolic stress present in the TME. Pancreatic cancer is characterized by a highly hypoxic and metabolically altered TME with increased lactate accumulation, making it a suitable model for investigating interactions between metabolic adaptation and LPA receptor-mediated signaling [13, 16].

In cholangiocarcinoma cells, short-term lactic acid treatment inhibited cell growth, whereas cell motility was enhanced following long-term lactic acid exposure [17]. In this study, we investigated the roles of LPA receptors in malignant behavior of pancreatic cancer PANC-1 cells, by generating long-term lactic acid-treated cells derived from parental PANC-1 cells. Using this model, we aimed to clarify whether prolonged exposure to lactic acid alters LPA receptor-mediated signaling and thereby influences malignant cellular functions of pancreatic cancer cells.

Materials and methods

Chemicals

Lactic acid was obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). LPA (1-oleoyl-2-hydroxy-sn-glycero-3-phosphate, 18:1) was purchased from Avanti Polar Lipid (Alabaster, AL). AM966, (2 S)-OMPT, AZD3965, and galloflavin were obtained from Cayman Chemical Company (Ann Arbor, MI). GRI-977,143 and TC LPA5 4 were purchased from Tocris Bioscience (Bristol, UK).

Cell culture and treatment

PANC-1 cells were obtained from ATCC (Manassas, VA). Cells were maintained in DMEM (Dulbecco’s Modified Eagle’s Medium) (cat. # 044–29765; FUJIFILM Wako) supplemented with 10% FBS in a humidified atmosphere containing 5% CO2 at 37 °C. In some experiments, cells were cultured in glucose-free DMEM (cat. # 042–32255; FUJIFILM Wako) supplemented with 5% charcoal-stripped FBS (Sigma-Aldrich, St. Louis, MO). For short-term experiments, cells were treated with lactic acid (1 or 5 mM) every 24 h for 2 days. Long-term lactic acid-treated cells were generated by stepwise exposure to lactic acid at concentrations ranging from 0.1 to 1 or 5 mM for approximately 3 months.

Cell viability and proliferation assays

Cells were seeded at a density of 5000 cells per well in 96-well plates and cultured in DMEM supplemented with 5% charcoal-stripped FBS. For cell viability assays, 5% charcoal-stripped FBS was used to minimize the influence of endogenous serum-derived factors on lactic acid responsiveness, whereas 10% FBS was used in proliferation assays to better reflect growth conditions that support active cellular proliferation [18]. Cells were treated with lactic acid (0, 1, 5, 10, 20, or 30 mM) every 24 h for 2 days. To evaluate adaptation to a lactic acid-rich environment, PANC-1 and long-term lactic acid-treated cells were also treated with lactic acid (0, 5, 10, 20, 30, or 50 mM) for 2 days. In some experiments, cells were cultured in serum-free DMEM for 2, 4, or 6 days. Cell viability was measured using the Cell Counting Kit-8 (CCK-8) (Dojindo, Kumamoto, Japan). The CCK-8 working solution was added to each well, followed by incubation at 37 °C for 1 h. Absorbance at 450 nm was then measured. For the cell proliferation assay, cells were suspended in DMEM containing 10% FBS and seeded at a density of 3000 cells per well in 96-well plates. Cell proliferation was evaluated using the CCK-8 on days 0, 1, and 2. Because viability and proliferation assays were performed under different serum conditions, direct quantitative comparison between these assays was not intended. All experiments were independently performed three times [13].

Quantitative real-time RT-PCR analysis

Total RNA was extracted using ISOGEN II (NIPPON GENE CO., LTD., Toyama, Japan) and reverse-transcribed into cDNA using PrimeScript RT Master Mix (Perfect Real Time) (TaKaRa Bio Inc., Shiga, Japan). Expression levels of LPA receptor genes were analyzed by quantitative real-time RT-PCR using TB Green Premix DimerEraser (TaKaRa Bio) and quantified with a Smart Cycler II System (TaKaRa Bio). The primer sequences used were as follows: human LPAR1 (GenBank accession number; BC030615) F: 5’-AGCAAGCTGGTGATGGGACT-3’, R: 5’-AGCGGCGGTTGACATAGATT-3’; human LPAR2 (GenBank accession number; BC025695) F: 5’-CTACCTGCTCGGCAATCTGG-3’, R: 5’-CAGGAACCAGCCCTCAAGTG-3’; human LPAR3 (GenBank accession number; BC126268) F: 5’-TGGCCTTCCTCATCATGGTT-3’, R: 5’-GCGGAGACAAGACGTTGGTT-3’; LPAR5 (GenBank accession number; BC072394) F: 5’- ACACATGCCATTGCGCTGTC-3’, R: 5’- TGCCACCCAAAGGTCCAAGT-3’; human monocarboxylate transporter 1 (MCT1) (GenBank accession number; BC026317) F: 5’-GCCTTCGGGTGGCTCAGCTC-3’, R: 5’-GGTCACCAATCCCACAGCGC-3’; human MCT4 (GenBank accession number; BC112269) F: 5’-GCCCAAAGAGCCACAGCCTG-3’, R: 5’-CTCCACCTCCCGCAAGTCCA-3’; and human GAPDH (GenBank accession number; BC026907) F: 5’-AGTCCATGCCATCACTGCCA-3’, R: 5’-CAGGTCCACCACTGACACGT-3’. All analyses were performed in three independent experiments.

Cell motility assay

A Cell Culture Insert (upper chamber) with 8.0 μm pore size (BD Falcon, Franklin Lakes, NJ) was placed in 24-well plates (lower chamber) containing 5% charcoal-stripped FBS-DMEM with or without LPA (1 µM). Prior to the cell motility assay, cells were treated with lactic acid (5 mM) for 30 min, then suspended in serum-free DMEM and seeded into each insert, followed by incubation for 16 h. To evaluate LPA receptor-mediated signaling, cells were treated with (2 S)-OMPT (1 µM) or GRI-977,143 (1 µM) instead of LPA under the same conditions. In some experiments, cells were pretreated with AM966 (1 µM) or TC LPA5 4 (1 µM) for 30 min before incubation in 5% charcoal-stripped FBS-DMEM containing LPA (1 µM). Following Giemsa staining, migrated cells on the exterior side of the inserts (upper chamber) were counted. Each experiment was independently repeated three times [13].

Cell growth assay with LPA and LPA receptor-mediated signaling modulators

Cells were seeded at a density of 3000 cells per well in 96-well plates and cultured in 5% charcoal-stripped FBS-DMEM. Cells were treated with LPA, GRI-977,143, or (2 S)-OMPT at concentrations of 0, 1, or 5 µM, added every 24 h for 2 days. In some experiments, cells were pretreated with AM966 or TC LPA5 4 at concentrations of 0, 1, or 5 µM for 30 min before stimulation with 1 µM LPA. This treatment was repeated every 24 h for 2 days. Cell growth was determined using the CCK-8. All experiments were independently performed three times [14].

Lactate assay

Lactate production was quantified using a Lactate Assay Kit-WST (Dojindo) according to the manufacturer’s instructions. Cells were seeded at a density of 5,000 cells per well in 96-well plates and maintained in DMEM supplemented with 5% charcoal-stripped FBS. After 24 h of culture, the working solution was added to each well and incubated at 37 °C for 30 min. Absorbance of the conditioned media was measured at 450 nm. For each experimental condition, four wells per cell type were analyzed, and the entire experiment was independently repeated three times.

Statistical analysis

Statistical analyses were performed using analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple group comparisons. Comparisons between two groups were evaluated using Student’s t-test. A p-value of less than 0.01 was considered statistically significant. Data are presented as the mean ± SD from at least three independent biological replicates.

Results

Effects of short-term lactic acid treatment on cellular responses of PANC-1 cells

To determine how short-term exposure to lactic acid affects proliferation, motility, and LPA receptor expression in PANC-1 cells, cells were treated with lactic acid for 48 h. Short-term exposure to 1 and 5 mM lactic acid did not reduce cell viability, whereas 10–30 mM significantly suppressed viability (Fig. 1A). Cell motility increased at 1 mM lactic acid, reached a maximum at 5 mM, and declined at higher concentrations (Fig. 1B). PANC-1 cell proliferation was significantly enhanced at 1 and 5 mM, with 5 mM inducing a greater increase (Fig. 1C). LPAR1 expression increased in a dose-dependent manner at 1 and 5 mM, while LPAR2 and LPAR3 expression remained unchanged. In contrast, LPAR5 expression decreased dose-dependently (Fig. 1D). PANC-1 cell motile was significantly stimulated by 1 and 5 mM lactic acid. Under these conditions, LPA further enhanced motility, whereas LPA alone did not alter motility (Fig. 1E). These findings indicate that short-term lactic acid exposure enhances proliferation and motility of PANC-1 cells, accompanied by increased LPAR1 and reduced LPAR5 expression, suggesting altered LPA receptor signaling responsiveness.

Fig. 1.

Fig. 1

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Cellular responses to lactic acid in PANC-1 cells. A Cell viability assay. Cells were cultured in 5% charcoal-stripped FBS-DMEM and treated with lactic acid (0, 1, 5, 10, 20, and 30 mM) for 2 days. Data are presented as means ± SD from three independent experiments. *; p < 0.01 vs. PANC-1 cells without lactic acid treatment. **; p < 0.001 vs. PANC-1 cells without lactic acid treatment. B Cell motility assay. Cells were suspended in serum-free DMEM and seeded inside the insert (upper chamber). The insert was placed in a 24-well plate (lower chamber) containing 5% charcoal-stripped FBS-DMEM. Cells were treated with lactic acid (0, 1, 5, 10, or 20 mM) and cultured for 16 h. Data are presented as means ± SD from three independent experiments. *; p < 0.01 vs. untreated PANC-1 cells. **; p < 0.001 vs. untreated PANC-1 cells. #; p < 0.01 between the indicated groups. C Cell proliferation assay. Cells were cultured in 10% FBS-DMEM and treated with lactic acid (0, 1, or 5 mM) every 24 h for 2 days. Cell proliferation is expressed as a percentage relative to the cell number on day 0. Assays were performed in triplicate. *; p < 0.01 vs. control. #; p < 0.01 vs. LA 1mM. control, untreated PANC-1 cells. LA 1 mM, PANC-1 cells treated with 1 mM lactic acid. LA 5 mM, PANC-1 cells treated with 5 mM lactic acid. D LPA receptor expression determined by quantitative real-time RT-PCR analysis. Data are presented as means ± SD from three independent experiments. *; p < 0.01 vs. control. #; p < 0.01 between the indicated groups. E Effects of LPA on cell motility. Cells were seeded inside the insert (upper chamber), and the insert was placed in a 24-well plate (lower chamber) containing 5% charcoal-stripped FBS-DMEM with or without LPA (1 µM). Cells were treated with lactic acid (0, 1, or 5 mM) and cultured for 16 h. Data are presented as means ± SD from three independent experiments. *; p < 0.01 vs. control. †; p < 0.01 vs. LPA-treated control. #; p < 0.01 between the indicated groups

Cell proliferation and LPA receptor expression in long-term lactic acid-treated PANC-1 cells

The impact of prolonged lactic acid exposure on proliferative capacity and LPA receptor expression was examined. PANC-LA1 and PANC-LA5 cells were generated by culturing PANC-1 cells with 1 or 5 mM lactic acid, respectively, for approximately 3 months (Fig. 2A). Both derivatives exhibited significantly higher proliferation than parental PANC-1 cells, with PANC-LA5 cells showing the greatest increase (Fig. 2B). LPAR1, LPAR3, and LPAR5 expression levels were elevated, whereas LPAR2 expression was reduced. Expression levels were generally higher in PANC-LA1 cells than in PANC-LA5 cells (Fig. 2C). These results indicate that long-term lactic acid exposure enhances proliferative capacity and alters LPA receptor expression profiles in PANC-1 cells.

Fig. 2.

Fig. 2

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Cell proliferation and LPA receptor expression in long-term lactic acid-treated PANC-1 cells. A Morphology of PANC-1 and long-term lactic acid-treated cells in 10% FBS-DMEM. PANC-LA1, PANC-1 cells treated with 1 mM lactic acid. PANC-LA5, PANC-1 cells treated with 5 mM lactic acid. Scale bars, 100 μm. B Cell proliferation assay. Cells were cultured in 10% FBS-DMEM. Data are expressed as percentages relative to day 0. Experiments were performed in triplicate. *; p < 0.01 vs. PANC-1 cells. **; p < 0.001 vs. PANC-1 cells. ##; p < 0.001 vs. PANC-LA1 cells. C LPA receptor expression determined by quantitative real-time RT-PCR analysis. Data are presented as the mean of three independent experiments. *; p < 0.01 vs. PANC-1 cells. #; p < 0.01 between the indicated groups

Cell growth and motility in long-term lactic acid-treated PANC-1 cells

We examined whether long-term lactic acid adaptation alters cellular responses to LPA. LPA (5 µM) reduced growth of parental PANC-1 cells. In contrast, PANC-LA5 cells exhibited dose-dependent growth stimulation in response to LPA, whereas PANC-LA1 cells showed no significant change (Fig. 3A). Cell motility was significantly higher in PANC-LA1 and PANC-LA5 cells than in PANC-1 cells, with PANC-LA1 cells showing greater motility than PANC-LA5 cells. LPA stimulated motility in both long-term treated cell lines, whereas PANC-1 cells remained unresponsive (Fig. 3B). These findings indicate that long-term lactic acid adaptation results in distinct phenotypes: PANC-LA5 cells exhibit enhanced LPA-responsive proliferation, while PANC-LA1 cells display increased motility. Cell viability was also examined following exposure to 0–50 mM lactic acid for 2 days. PANC-LA1 and PANC-LA5 cells exhibited significantly higher viability than parental PANC-1 cells at 10–50 mM lactic acid. In addition, PANC-LA5 cells showed significantly higher viability than PANC-LA1 cells within this concentration range (Fig. 3C). These results demonstrate that long-term lactic acid exposure enhances tolerance to lactic acid-rich conditions, with the greatest adaptation observed in PANC-LA5 cells.

Fig. 3.

Fig. 3

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Cellular functions of long-term lactic acid-treated PANC-1 cells. A Cell growth in response to LPA. Cells were cultured for 2 days with LPA (0, 1, or 5 µM). Data are presented as means ± SD from three independent experiments. **; p < 0.001 vs. LPA-untreated cells. #; p < 0.01 between the indicated groups. PANC-LA1, PANC-1 cells treated with 1 mM lactic acid. PANC-LA5, PANC-1 cells treated with 5 mM lactic acid. B Cell motility assay. Cells were seeded in the insert (upper chamber), and the insert was placed in a 24-well plate (lower chamber) containing 5% charcoal-stripped FBS-DMEM with or without LPA (1 µM). Data are presented as means ± SD from three independent experiments. *; p < 0.01 vs. LPA-untreated PANC-1 cells. **; p < 0.001 vs. LPA-untreated PANC-1 cells. ††; p < 0.001 vs. LPA-treated PANC-1 cells. #; p < 0.01 between the indicated groups. ##; p < 0.001 between the indicated groups. C Cell viability assay. Cells were cultured in 5% charcoal-stripped FBS-DMEM and treated with lactic acid (0, 5, 10, 20, 30, and 50 mM) for 2 days. Data are presented as means ± SD from three independent experiments. *; p < 0.01 vs. PANC-1 cells. **; p < 0.001 vs. PANC-1 cells. #; p < 0.01 vs. PANC-LA1 cells. ##; p < 0.001 vs. PANC-LA1 cells

Roles of LPA receptor signaling in cell growth and motility of long-term lactic acid-treated PANC-1 cells

Specific LPA receptor subtypes contributing to altered growth and motility were investigated using pharmacological modulators. Because LPA-responsive proliferation was more pronounced in PANC-LA5 cells and motility was greater in PANC-LA1 cells, PANC-LA5 cells were used to evaluate proliferation, whereas PANC-LA1 cells were used to assess motility. Accordingly, LPA co-treatment experiments were performed under conditions in which the respective phenotypic responses were most clearly observed. In PANC-LA5 cells, LPA-induced growth was suppressed by AM966 (LPA1 antagonist) and enhanced by TC LPA5 4 (LPA5 antagonist) (Fig. 4A). GRI-977,143 (LPA2 agonist) and (2 S)-OMPT (LPA3 agonist) increased cell growth (Fig. 4B). In PANC-LA1 cells, LPA-induced motility was inhibited by AM966 and enhanced by TC LPA5 4 (Fig. 4C). Similarly, GRI-977,143 reduced motility, while (2 S)-OMPT enhanced it (Fig. 4D). These findings suggest that LPA1 and LPA3 stimulate growth and motility, whereas LPA2 differentially regulates these processes by enhancing growth and suppressing motility, and LPA5 exerts inhibitory effects.

Fig. 4.

Fig. 4

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Roles of LPA receptor signaling in cell growth and motility of long-term lactic acid-treated PANC-1 cells. A Effects of LPA1 and LPA5 antagonists on cell growth. Cells were pre-incubated with AM966 or TC LPA5 4 prior to stimulation with LPA (1 µM) for 2 days. Data are presented as means ± SD from three independent experiments. *; p < 0.01 vs. TC LPA5 4-untreated cells. **; p < 0.001 vs. AM966- or TC LPA5 4-untreated cells. ##: p < 0.001 between the indicated groups. PANC-LA5, PANC-1 cells treated with 5 mM lactic acid. B Effects of LPA2 and LPA3 agonists on cell growth. Cells were treated with GRI-977,143 or (2 S)-OMPT for 2 days. Data are presented as means ± SD from three independent experiments. *; p < 0.01 vs. GRI-977,143- or (2 S)-OMPT-untreated cells. **; p < 0.001 vs. GRI-977,143- or (2 S)-OMPT-untreated cells. C Effects of LPA1 and LPA5 antagonists on cell motility. Cells were pre-incubated with AM966 or TC LPA5 4 prior to stimulation with LPA (1 µM). Data are presented as means ± SD from three independent experiments. *; p < 0.01 vs. control. #; p < 0.01 between the indicated groups. control, untreated cells. PANC-LA1, PANC-1 cells treated with 1 mM lactic acid. D Effects of LPA2 and LPA3 agonists on cell motility. Data are presented as means ± SD from three independent experiments. *; p < 0.01 vs. control. control, untreated cells

Effects of lactic acid metabolism on cell growth and motility of long-term lactic acid-treated PANC-1 cells

We examined whether metabolic reprogramming contributes to enhanced cellular functions in long-term lactic acid-treated cells. Extracellular lactate production was significantly lower in PANC-LA1 and PANC-LA5 cells than in PANC-1 cells, with the lowest levels observed in PANC-LA5 cells (Fig. 5A). MCT1 expression was elevated in both adapted cell lines, particularly in PANC-LA5 cells, whereas MCT4 expression remained unchanged (Fig. 5B). Under glucose-free conditions, viability of PANC-LA1 and PANC-LA5 cells was higher than that of PANC-1 cells. PANC-1 viability declined by day 2, whereas viability of PANC-LA1 and PANC-LA5 cells increased at this time point. The survival rate of PANC-LA5 cells exceeded that of PANC-LA1 cells under these conditions. After day 4, viability of both adapted cell lines decreased, although PANC-LA5 cells consistently exhibited higher survival than PANC-LA1 cells (Fig. 5C). In the presence of LPA, LPA-induced growth in PANC-LA5 cells was suppressed by AZD3965 (MCT1 inhibitor) and galloflavin (lactate dehydrogenase (LDH) inhibitor) (Fig. 5D). Both inhibitors also reduced motility of PANC-LA1 cells (Fig. 5E). These results indicate that long-term lactic acid exposure induces metabolic adaptation characterized by reduced lactate production and increased MCT1 expression, thereby supporting LPA-dependent growth and motility.

Fig. 5.

Fig. 5

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Effects of lactic acid on cellular functions of long-term lactic acid-treated PANC-1 cells. A Lactate production assay. Cells were maintained in DMEM supplemented with 5% charcoal-stripped FBS. Lactate levels were measured using the Lactate Assay Kit-WST. Data are presented as means ± SD from three independent experiments. *; p < 0.01 vs. PANC-1 cells. **; p < 0.001 vs. PANC-1 cells. #; p < 0.01 between the indicated groups. PANC-LA1, PANC-1 cells treated with 1 mM lactic acid. PANC-LA5, PANC-1 cells treated with 5 mM lactic acid. B MCT1 and MCT4 expression determined by quantitative real-time RT-PCR analysis. Data are presented as means ± SD from three independent experiments. *; p < 0.01 vs. PANC-1 cells. #; p < 0.01 between the indicated groups. C Cell viability under glucose-free conditions. cells were cultured in glucose-free DMEM supplemented with 5% charcoal-stripped FBS. Data are presented as means ± SD from three independent experiments. *; p < 0.01 vs. PANC-1 cells. **; p < 0.001 vs. PANC-1 cells. #; p < 0.01 vs. PANC-LA1 cells. ##; p < 0.001 vs. PANC-LA1 cells. D Effects of AZD3965 and galloflavin on cell growth. Cells were pre-incubated with AZD3965 (1 mM) or galloflavin (1 mM) for 30 min, followed by stimulation with LPA (1 µM) and culture for 2 days. Data are presented as means ± SD from three independent experiments. *; p < 0.01 vs. untreated cells. **; p < 0.001 vs. untreated cells. #: p < 0.01 between the indicated groups. ##: p < 0.001 between the indicated groups. E Effects of AZD3965 and galloflavin on cell motility. Cells were pre-incubated with AZD3965 (1 mM) or galloflavin (1 mM) and treated with LPA (1 µM). Data are presented as means ± SD from three independent experiments. **; p < 0.001 vs. untreated cells. ##: p < 0.001 between the indicated groups

Discussion

LPA receptors are divided into endothelial cell differentiation gene (Edg) (LPA1-LPA3) and non-Edg (LPA4-LPA6) families and exert cell type-dependent biological effects [7–9]. In PANC-1 cells, previous studies demonstrated that LPA1 and LPA3 promote motility, whereas LPA2 exerts inhibitory effects [19, 20]. Accordingly, this study focused on LPA1-LPA3 to further define differential roles in regulating proliferation and motility in long-term lactic acid-treated cells. In addition, LPA5 was examined based on evidence indicating an inhibitory role in cell growth and motility [21], enabling evaluation of both stimulatory and suppressive LPA receptor signaling. This classification provides a useful framework to interpret how distinct receptor subtypes contribute to heterogeneous cellular responses under metabolically altered conditions. Furthermore, examining both Edg and non-Edg receptors enables a more comprehensive understanding of how LPA signaling is reprogrammed following long-term lactic acid exposure.

To generate long-term lactic acid-treated cells, the concentration of lactic acid was first determined. Treatment with 5 mM lactic acid resulted in the highest motility and proliferation without reducing cell viability (Fig. 1A–C). PANC-1 cell motility induced by lactic acid at 1 and 5 mM was further stimulated by LPA, whereas LPA had no effect on untreated cells, accompanied by changes in LPAR1 and LPAR5 expression (Fig. 1D, E) [19, 21]. Because LPA can activate multiple LPA receptor subtypes, including LPA1-LPA6, the observed cellular responses are likely determined by the integrated balance of signaling from receptors with stimulatory and inhibitory functions rather than by a single receptor subtype. Although differences in ligand preference and receptor potency among LPA receptor subtypes have been reported, all LPA receptors can be activated by 1-acyl-LPA species, including the LPA used in the present study [22]. Therefore, the increased motility observed following LPA stimulation may be attributable to enhanced LPAR1 expression together with reduced LPAR5 expression, thereby shifting the balance toward promigratory signaling. This interpretation is consistent with the effects of the LPA1 antagonist AM966 and the LPA5 antagonist TC LPA5 4 on cell motility (Fig. 4C). Moreover, the differential effects observed with receptor-selective agonists and antagonists compared with LPA treatment may reflect the fact that LPA simultaneously activates multiple receptor subtypes, whereas pharmacological modulators selectively target individual receptors. Therefore, the proliferative and motility responses induced by LPA are likely to represent the net outcome of both positive and negative LPA receptor-mediated signals. Based on these findings, PANC-LA1 and PANC-LA5 cells were established by gradual exposure to 1 and 5 mM lactic acid, respectively. PANC-LA1 and PANC-LA5 cells showed elevated proliferation, similar to short-term lactic acid treatment (Figs. 1C and 2B). In contrast, LPAR1, LPAR3, and LPAR5 expression levels were increased, while LPAR2 expression was reduced (Fig. 2C). Notably, the expression patterns of LPA receptors differed not only between parental PANC-1 cells and long-term lactic acid-treated cells, but also between PANC-LA1 and PANC-LA5 cells. LPAR1 and LPAR3 expression levels were generally higher in PANC-LA1 cells, whereas proliferative responses were more pronounced in PANC-LA5 cells, suggesting that distinct receptor expression profiles induced by different lactic acid concentrations may contribute to the divergence between motility- and proliferation-associated phenotypes. LPA enhanced cell growth in PANC-LA5 cells, whereas it had no significant effect on PANC-LA1 cells, in contrast to the inhibitory effect observed in parental PANC-1 cells, indicating increased responsiveness to LPA following long-term lactic acid exposure (Fig. 3A). These findings suggest that long-term lactic acid treatment differentially reprograms LPA receptor-mediated signaling depending on the metabolic conditions used for adaptation. Although LPAR1, LPAR3, and LPAR5 expression levels were generally higher in PANC-LA1 cells than in PANC-LA5 cells, LPA-induced proliferation was more evident in PANC-LA5 cells. Because LPA functions as a ligand for multiple LPA receptor subtypes, the resulting phenotype may reflect the integrated balance between stimulatory and inhibitory receptor-mediated signaling rather than the expression level of an individual receptor alone. To further evaluate receptor involvement, pharmacological modulators were applied. AM966, TC LPA5 4, GRI-977,143, and (2 S)-OMPT were used as pharmacological modulators for LPA1, LPA5, LPA2, and LPA3, respectively [23–26]. PANC-LA5 cell growth was inhibited by the LPA1 antagonist AM966 and stimulated by the LPA5 antagonist TC LPA5 4. Moreover, GRI-977,143 and (2 S)-OMPT, agonists of LPA2 and LPA3, respectively, also enhanced cell growth (Fig. 4A, B). These results indicate that LPA1-LPA3 promote, whereas LPA5 suppresses, PANC-LA5 cell growth, consistent with receptor expression changes induced by long-term lactic acid exposure. The observation that proliferation was enhanced by selective modulation of multiple LPA receptor subtypes suggests that individual receptors make distinct contributions to the overall proliferative response. However, the present data do not allow quantitative determination of the relative contribution of each receptor subtype. Rather, the proliferative effects observed following LPA stimulation are likely to reflect the integrated balance of signaling among multiple LPA receptors. Among these receptors, LPA1 and LPA3 appear to play central roles in mediating the enhanced proliferative and motility-associated phenotypes induced by long-term lactic acid exposure. In addition, these findings imply that long-term lactic acid exposure alters the functional balance among LPA receptor subtypes, thereby contributing to the increased responsiveness to LPA observed in PANC-LA5 cells. Proliferative activity was more pronounced following 5 mM lactic acid exposure, whereas motility was more evident after 1 mM exposure. This concentration-dependent divergence indicates that different levels of long-term lactic acid exposure are associated with distinct phenotypic responses and LPA receptor expression profiles. Consistent with these observations, both PANC-LA1 and PANC-LA5 cells exhibited greater tolerance to exogenous lactic acid than parental PANC-1 cells, and PANC-LA5 cells showed the highest viability under high-lactate conditions (Fig. 3C). These findings confirm that long-term lactic acid exposure induces adaptation to a lactic acid-rich environment and suggest that adaptation is more pronounced in PANC-LA5 cells, which were established under the higher lactic acid concentration.

Previous studies demonstrated that LPA1 and LPA3 promote motility in PANC-1 cells, whereas LPA2 and LPA5 exert inhibitory effects [13, 19, 21]. In the present study, PANC-1 cell motility was increased by long-term lactic acid exposure and further enhanced by LPA (Fig. 3B). In PANC-LA1 cells, motility was reduced by AM966 and GRI-977,143 and enhanced by (2 S)-OMPT and TC LPA5 4 (Fig. 4C, D). Thus, pharmacological inhibition of LPA5 increased both cell growth and motility, supporting an inhibitory role of endogenous LPA5 in regulating these cellular functions. Although LPAR5 expression was increased following long-term lactic acid exposure, the overall phenotype is likely determined by the integrated balance of signaling among multiple LPA receptor subtypes rather than by the expression level of a single receptor. Taken together, these results indicate that motility in long-term lactic acid-treated cells is associated with increased LPAR1 and LPAR3 expression and decreased LPAR2 expression. These findings support a model in which LPA1 and LPA3 promote proliferation and motility, whereas LPA2 and LPA5 exert inhibitory or counterbalancing effects. Consistent with these findings, our previous study demonstrated that shRNA-mediated knockdown of either LPA1 or LPA3 similarly reduced the motility, invasive ability, and colony-forming ability of PANC-1 cells [19]. These results support the involvement of both receptors in malignant phenotypes. However, the relative contribution of each receptor under long-term lactic acid exposure remains to be clarified by further genetic studies. Consequently, the cellular responses to LPA are likely determined by the net balance among multiple LPA receptor-mediated signals. Importantly, these observations parallel the changes seen in cell growth responses, further supporting the idea that long-term lactic acid exposure enhances overall cellular responsiveness to LPA signaling. In this context, both proliferative and motility-related processes appear to become more sensitive to LPA stimulation, although the extent of this enhancement varies depending on the specific cellular phenotype and receptor subtype involved.

We next investigated the roles of lactic acid in modulating cellular functions through LPA receptor signaling in long-term lactic acid-treated cells. Lactic acid is transported across the cellular membrane via MCTs [27]. MCTs are important for nutrient transport, metabolic regulation, and intracellular pH balance [28]. Among them, MCT1 and MCT4 cooperate to regulate proton gradients through lactate transport, contributing to intracellular alkalinization and extracellular acidification [5]. MCT1 expression was increased in long-term lactic acid-treated cells, whereas no change in MCT4 expression was observed (Fig. 5A, B). In the presence of LPA, cell growth and motility of long-term lactic acid-treated cells were suppressed by the MCT1 inhibitor AZD3965 [29]. LDH, which catalyzes the conversion of pyruvate to lactate, also contributed to these effects, as inhibition by galloflavin reduced growth and motility (Fig. 5D, E) [30, 31]. Furthermore, long-term lactic acid-treated cells exhibited higher viability under glucose-free conditions compared with PANC-1 cells (Fig. 5C). These findings demonstrate that long-term lactic acid exposure alters metabolic characteristics of PANC-1 cells and contributes to changes in cellular functions associated with LPA receptor signaling. Increased MCT1 expression suggests enhanced lactate transport activity, which may facilitate maintenance of intracellular pH and support metabolic adaptation under acidic conditions. The suppression of cell growth and motility by AZD3965 indicates that lactate transport via MCT1 is required for these responses under LPA stimulation. In addition, inhibition of LDH activity reduced growth and motility, indicating that intracellular lactate production also contributes to these effects. These findings suggest that lactate transport and production contribute to the enhanced cellular responses to LPA observed after long-term lactic acid exposure. Because the effects of AZD3965 and galloflavin were observed under LPA-stimulated conditions, MCT1-mediated lactate metabolism appears to function in concert with LPA receptor signaling rather than acting independently. Accordingly, lactate transport and utilization may contribute to the cellular responses induced by LPA in long-term lactic acid-treated cells. The increased survival under glucose-free conditions further supports the acquisition of an alternative metabolic state that sustains cellular functions under nutrient limitation. These observations indicate that long-term lactic acid-treated cells acquire metabolic characteristics associated with sustained cellular activity under nutrient-limited conditions. Collectively, these observations indicate that long-term lactic acid exposure modifies metabolic pathways involving lactate transport and production, thereby influencing LPA receptor-mediated cellular functions in PANC-1 cells (Fig. 6).

Fig. 6.

Fig. 6

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Schematic summary of cellular functions in long-term lactic acid-treated PANC-1 cells. A Effects of AZD3965 (MCT1 inhibitor) and galloflavin (LDH inhibitor) on cell growth and motility. MCT1, monocarboxylate transporter 1; MCT4, monocarboxylate transporter 4; LDH, lactate dehydrogenase; GLUT, glucose transporter. B Effects of LPA receptor signaling on cellular functions. PANC-LA1, PANC-1 cells treated with 1 mM lactic acid. PANC-LA5, PANC-1 cells treated with 5 mM lactic acid. ↑ or ↓, p < 0.01 vs. PANC-1 cells; ↑↑ or ↓↓, p < 0.001 vs. PANC-1 cells

Lactic acid functions not only an energy source for cancer cells but also as a regulator of cellular signaling pathways, that influence cancer cell behavior and therapeutic response [6]. Overexpression of MCT1 and MCT4 has been reported in several cancer types, including pancreatic cancer [32], and elevated serum LDH levels are associated with poor prognosis [30]. In this context, lactate metabolism represents a potential therapeutic target. Indeed, inhibition of MCT and LDH suppresses cancer cell activity [5]. The present study demonstrates that long-term lactic acid exposure enhances LPA-dependent growth and motility in PANC-1 cells, whereas inhibition of MCT1 and LDH reduces these effects. Notably, the shift from an inhibitory response to LPA in parental cells to an enhanced growth response in PANC-LA5 cells indicates a functional reprogramming of LPA receptor signaling during long-term lactic acid adaptation. This reprogramming may represent a key mechanism by which cancer cells adjust to metabolic stress and acquire more aggressive phenotypes. Thus, metabolic adaptation and LPA receptor signaling appear to be closely interconnected processes that cooperatively regulate cellular functions under conditions of prolonged lactic acid exposure. However, the present analysis was limited to mRNA expression levels. In addition, we did not examine whether treatment with receptor-selective agonists or antagonists altered the expression of LPA receptor isoforms. Therefore, compensatory regulation among LPA receptor subtypes cannot be excluded and should be investigated in future studies. Further investigation at the protein level, as well as functional validation in vivo, will be required to fully elucidate the mechanisms underlying this increased responsiveness to LPA and its potential clinical relevance.

Author contributions

M.Y. and M. Takai performed all experiments and analyzed data. N.S., E.I., N.Y., M.Tamura, S.N. and Y.K. assisted in cell culture experiments. T.T. designed this study and edited the manuscript. All authors reviewed the results and approved the manuscript.

Funding

This work was supported by Japan Society for the Promotion of Science (Grant Numbers 21K07109).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Mao Yamamoto and Miwa Takai equally contributed to this work.

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