Limits...
Stromal cells positively and negatively modulate the growth of cancer cells: stimulation via the PGE2-TNFα-IL-6 pathway and inhibition via secreted GAPDH-E-cadherin interaction.

Kawada M, Inoue H, Ohba S, Yoshida J, Masuda T, Yamasaki M, Usami I, Sakamoto S, Abe H, Watanabe T, Yamori T, Shibasaki M, Nomoto A - PLoS ONE (2015)

Bottom Line: Furthermore, we found that stromal cells secreted glyceraldehyde 3-phosphate dehydrogenase (GAPDH).Extracellular GAPDH, or its N-terminal domain, inhibited gastric cancer cell growth, a finding confirmed in other cell systems.These results demonstrate that stromal cells could regulate cancer cell growth through the balance of these secreted factors.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation, Numazu-shi, Shizuoka, Japan.

ABSTRACT
Fibroblast-like stromal cells modulate cancer cells through secreted factors and adhesion, but those factors are not fully understood. Here, we have identified critical stromal factors that modulate cancer growth positively and negatively. Using a cell co-culture system, we found that gastric stromal cells secreted IL-6 as a growth and survival factor for gastric cancer cells. Moreover, gastric cancer cells secreted PGE2 and TNFα that stimulated IL-6 secretion by the stromal cells. Furthermore, we found that stromal cells secreted glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Extracellular GAPDH, or its N-terminal domain, inhibited gastric cancer cell growth, a finding confirmed in other cell systems. GAPDH bound to E-cadherin and downregulated the mTOR-p70S6 kinase pathway. These results demonstrate that stromal cells could regulate cancer cell growth through the balance of these secreted factors. We propose that negative regulation of cancer growth using GAPDH could be a new anti-cancer strategy.

No MeSH data available.


Related in: MedlinePlus

Gastric stromal cells secreted GAPDH and suppressed the growth of gastric cancer cells.(A) Concentrated Hs738 CM prepared by culturing with or without 10 μM MEK inhibitor I was analyzed by Western blot (upper left). Hs738 cells were cultured with MEK inhibitor I for 2 days and the cultured supernatant was collected. GAPDH and enolase in the culture supernatant (ex) and the cell lysates (in) was analyzed by Western blot (upper right). GAPDH enzyme activity was examined in the cultured supernatant (lower). The values are means ± s.d. (n = 3). (B) MKN-7 and MKN-74 cells were cultured with various amounts of GAPDH for 3 days. Cell growth was determined using MTT. The values are means ± s.d. (n = 3). (C) Gastric cancer cells were cultured with or without human erythrocyte GAPDH at 5 U/mL for 1 day. The cell lysates were analyzed by Western blotting with anti-phospho-Ser/Thr antibody (9624) and the indicated antibodies. An arrowhead indicates the position of RPS6. (D) MKN-7 cells were cultured with human recombinant deletion mutants of GAPDH for 3 days. Cell growth was determined using MTT. The values are means ± s.d. (n = 3). Cell growth is expressed as a percentage of the value without GAPDH in each culture condition.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4364666&req=5

pone.0119415.g006: Gastric stromal cells secreted GAPDH and suppressed the growth of gastric cancer cells.(A) Concentrated Hs738 CM prepared by culturing with or without 10 μM MEK inhibitor I was analyzed by Western blot (upper left). Hs738 cells were cultured with MEK inhibitor I for 2 days and the cultured supernatant was collected. GAPDH and enolase in the culture supernatant (ex) and the cell lysates (in) was analyzed by Western blot (upper right). GAPDH enzyme activity was examined in the cultured supernatant (lower). The values are means ± s.d. (n = 3). (B) MKN-7 and MKN-74 cells were cultured with various amounts of GAPDH for 3 days. Cell growth was determined using MTT. The values are means ± s.d. (n = 3). (C) Gastric cancer cells were cultured with or without human erythrocyte GAPDH at 5 U/mL for 1 day. The cell lysates were analyzed by Western blotting with anti-phospho-Ser/Thr antibody (9624) and the indicated antibodies. An arrowhead indicates the position of RPS6. (D) MKN-7 cells were cultured with human recombinant deletion mutants of GAPDH for 3 days. Cell growth was determined using MTT. The values are means ± s.d. (n = 3). Cell growth is expressed as a percentage of the value without GAPDH in each culture condition.

Mentions: Hs738 CM downregulated phosphorylation of RPS6 and this inhibitory effect was more pronounced in MEK inhibitor I-treated CM suggesting that Hs738 CM contained growth-inhibitory activities (Fig. 2, S3C Fig., and S15 Fig.). To analyze those factors, we concentrated CM from Hs738 cells pretreated with MEK inhibitor I and purified the proteins (S15 Fig.). Proteomic analysis of gel filtrated fractions revealed that the candidate proteins were enolase, PAI-1, and GAPDH (S15 Fig.). Western blotting revealed that one of the growth inhibitory factors might be GAPDH. In fact, MEK inhibitor I increased extracellular secretion of GAPDH without affecting the intracellular levels of GAPDH and the extracellular enolase (Fig. 6A). The amount of secreted GAPDH was higher in Hs738 cells than gastric cancer cell lines (S16 Fig.). Furthermore, other stromal cells also secreted GAPDH (S16 Fig.). Although GAPDH is reported to be secreted and to change cell morphology [34], its growth inhibitory activity has not been reported.


Stromal cells positively and negatively modulate the growth of cancer cells: stimulation via the PGE2-TNFα-IL-6 pathway and inhibition via secreted GAPDH-E-cadherin interaction.

Kawada M, Inoue H, Ohba S, Yoshida J, Masuda T, Yamasaki M, Usami I, Sakamoto S, Abe H, Watanabe T, Yamori T, Shibasaki M, Nomoto A - PLoS ONE (2015)

Gastric stromal cells secreted GAPDH and suppressed the growth of gastric cancer cells.(A) Concentrated Hs738 CM prepared by culturing with or without 10 μM MEK inhibitor I was analyzed by Western blot (upper left). Hs738 cells were cultured with MEK inhibitor I for 2 days and the cultured supernatant was collected. GAPDH and enolase in the culture supernatant (ex) and the cell lysates (in) was analyzed by Western blot (upper right). GAPDH enzyme activity was examined in the cultured supernatant (lower). The values are means ± s.d. (n = 3). (B) MKN-7 and MKN-74 cells were cultured with various amounts of GAPDH for 3 days. Cell growth was determined using MTT. The values are means ± s.d. (n = 3). (C) Gastric cancer cells were cultured with or without human erythrocyte GAPDH at 5 U/mL for 1 day. The cell lysates were analyzed by Western blotting with anti-phospho-Ser/Thr antibody (9624) and the indicated antibodies. An arrowhead indicates the position of RPS6. (D) MKN-7 cells were cultured with human recombinant deletion mutants of GAPDH for 3 days. Cell growth was determined using MTT. The values are means ± s.d. (n = 3). Cell growth is expressed as a percentage of the value without GAPDH in each culture condition.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4364666&req=5

pone.0119415.g006: Gastric stromal cells secreted GAPDH and suppressed the growth of gastric cancer cells.(A) Concentrated Hs738 CM prepared by culturing with or without 10 μM MEK inhibitor I was analyzed by Western blot (upper left). Hs738 cells were cultured with MEK inhibitor I for 2 days and the cultured supernatant was collected. GAPDH and enolase in the culture supernatant (ex) and the cell lysates (in) was analyzed by Western blot (upper right). GAPDH enzyme activity was examined in the cultured supernatant (lower). The values are means ± s.d. (n = 3). (B) MKN-7 and MKN-74 cells were cultured with various amounts of GAPDH for 3 days. Cell growth was determined using MTT. The values are means ± s.d. (n = 3). (C) Gastric cancer cells were cultured with or without human erythrocyte GAPDH at 5 U/mL for 1 day. The cell lysates were analyzed by Western blotting with anti-phospho-Ser/Thr antibody (9624) and the indicated antibodies. An arrowhead indicates the position of RPS6. (D) MKN-7 cells were cultured with human recombinant deletion mutants of GAPDH for 3 days. Cell growth was determined using MTT. The values are means ± s.d. (n = 3). Cell growth is expressed as a percentage of the value without GAPDH in each culture condition.
Mentions: Hs738 CM downregulated phosphorylation of RPS6 and this inhibitory effect was more pronounced in MEK inhibitor I-treated CM suggesting that Hs738 CM contained growth-inhibitory activities (Fig. 2, S3C Fig., and S15 Fig.). To analyze those factors, we concentrated CM from Hs738 cells pretreated with MEK inhibitor I and purified the proteins (S15 Fig.). Proteomic analysis of gel filtrated fractions revealed that the candidate proteins were enolase, PAI-1, and GAPDH (S15 Fig.). Western blotting revealed that one of the growth inhibitory factors might be GAPDH. In fact, MEK inhibitor I increased extracellular secretion of GAPDH without affecting the intracellular levels of GAPDH and the extracellular enolase (Fig. 6A). The amount of secreted GAPDH was higher in Hs738 cells than gastric cancer cell lines (S16 Fig.). Furthermore, other stromal cells also secreted GAPDH (S16 Fig.). Although GAPDH is reported to be secreted and to change cell morphology [34], its growth inhibitory activity has not been reported.

Bottom Line: Furthermore, we found that stromal cells secreted glyceraldehyde 3-phosphate dehydrogenase (GAPDH).Extracellular GAPDH, or its N-terminal domain, inhibited gastric cancer cell growth, a finding confirmed in other cell systems.These results demonstrate that stromal cells could regulate cancer cell growth through the balance of these secreted factors.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation, Numazu-shi, Shizuoka, Japan.

ABSTRACT
Fibroblast-like stromal cells modulate cancer cells through secreted factors and adhesion, but those factors are not fully understood. Here, we have identified critical stromal factors that modulate cancer growth positively and negatively. Using a cell co-culture system, we found that gastric stromal cells secreted IL-6 as a growth and survival factor for gastric cancer cells. Moreover, gastric cancer cells secreted PGE2 and TNFα that stimulated IL-6 secretion by the stromal cells. Furthermore, we found that stromal cells secreted glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Extracellular GAPDH, or its N-terminal domain, inhibited gastric cancer cell growth, a finding confirmed in other cell systems. GAPDH bound to E-cadherin and downregulated the mTOR-p70S6 kinase pathway. These results demonstrate that stromal cells could regulate cancer cell growth through the balance of these secreted factors. We propose that negative regulation of cancer growth using GAPDH could be a new anti-cancer strategy.

No MeSH data available.


Related in: MedlinePlus