Limits...
Protein kinase C-δ inactivation inhibits the proliferation and survival of cancer stem cells in culture and in vivo.

Chen Z, Forman LW, Williams RM, Faller DV - BMC Cancer (2014)

Bottom Line: Inhibition of PKCδ by specific genetic strategies (shRNA) or by novel small molecule inhibitors is growth inhibitory and cytotoxic to multiple types of human CSCs in culture.PKCδ inhibition efficiently prevents tumor sphere outgrowth from tumor cell cultures, with exposure times as short as six hours.Small-molecule PKCδ inhibitors also inhibit human CSC growth in vivo in a mouse xenograft model.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Center, Boston University School of Medicine, K-712C, 72 E, Concord St,, Boston, MA 02118, USA. dfaller@bu.edu.

ABSTRACT

Background: A subpopulation of tumor cells with distinct stem-like properties (cancer stem-like cells, CSCs) may be responsible for tumor initiation, invasive growth, and possibly dissemination to distant organ sites. CSCs exhibit a spectrum of biological, biochemical, and molecular features that are consistent with a stem-like phenotype, including growth as non-adherent spheres (clonogenic potential), ability to form a new tumor in xenograft assays, unlimited self-renewal, and the capacity for multipotency and lineage-specific differentiation. PKCδ is a novel class serine/threonine kinase of the PKC family, and functions in a number of cellular activities including cell proliferation, survival or apoptosis. PKCδ has previously been validated as a synthetic lethal target in cancer cells of multiple types with aberrant activation of Ras signaling, using both genetic (shRNA and dominant-negative PKCδ mutants) and small molecule inhibitors. In contrast, PKCδ is not required for the proliferation or survival of normal cells, suggesting the potential tumor-specificity of a PKCδ-targeted approach.

Methods: shRNA knockdown was used validate PKCδ as a target in primary cancer stem cell lines and stem-like cells derived from human tumor cell lines, including breast, pancreatic, prostate and melanoma tumor cells. Novel and potent small molecule PKCδ inhibitors were employed in assays monitoring apoptosis, proliferation and clonogenic capacity of these cancer stem-like populations. Significant differences among data sets were determined using two-tailed Student's t tests or ANOVA.

Results: We demonstrate that CSC-like populations derived from multiple types of human primary tumors, from human cancer cell lines, and from transformed human cells, require PKCδ activity and are susceptible to agents which deplete PKCδ protein or activity. Inhibition of PKCδ by specific genetic strategies (shRNA) or by novel small molecule inhibitors is growth inhibitory and cytotoxic to multiple types of human CSCs in culture. PKCδ inhibition efficiently prevents tumor sphere outgrowth from tumor cell cultures, with exposure times as short as six hours. Small-molecule PKCδ inhibitors also inhibit human CSC growth in vivo in a mouse xenograft model.

Conclusions: These findings suggest that the novel PKC isozyme PKCδ may represent a new molecular target for cancer stem cell populations.

Show MeSH

Related in: MedlinePlus

Effects of a 3rd generation small molecule PKCδ inhibitor on human pancreatic cancer stem cell cultures. PCSC cells were grown to 80% confluence in 96-well plates and then exposed to BJE6-106 at concentrations ranging from 0.1 to 20 μM, or to rottlerin at concentrations ranging from 1 to 20 μM. The corresponding equivalent volume of solvent (DMSO) was used as a vehicle control (Vehicle). After 48 and 72 hr of exposure, cell mass was evaluated by MTT assay. Control values were normalized to 100%. Error bars represent SEM. p values for comparison between vehicle and rottlerin effects on cell number at 48 hr reached significance at 1 μM, and for BJE6-106 at 0.1 μM (p ≤ 0.02), and remained significant at the 72 hr time point.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC3927586&req=5

Figure 3: Effects of a 3rd generation small molecule PKCδ inhibitor on human pancreatic cancer stem cell cultures. PCSC cells were grown to 80% confluence in 96-well plates and then exposed to BJE6-106 at concentrations ranging from 0.1 to 20 μM, or to rottlerin at concentrations ranging from 1 to 20 μM. The corresponding equivalent volume of solvent (DMSO) was used as a vehicle control (Vehicle). After 48 and 72 hr of exposure, cell mass was evaluated by MTT assay. Control values were normalized to 100%. Error bars represent SEM. p values for comparison between vehicle and rottlerin effects on cell number at 48 hr reached significance at 1 μM, and for BJE6-106 at 0.1 μM (p ≤ 0.02), and remained significant at the 72 hr time point.

Mentions: The activity of the 3rd generation PKCδ inhibitor BJE6-106 on the growth of PCSC cells in culture was compared to rottlerin. BJE6-106 inhibited the growth of PCSC cultures at concentrations as low as 0.1 μM, and had an (in culture) IC50 of approximately 0.5 μM at 48 hr (Figure 3). In contrast, rottlerin produced no significant inhibitory activity at 0.5 μM, and displayed an IC50 at 48 hr of approximately 3 μM. LDH release assays also showed greater than 10-fold increases in potency for BJE6-106 compared to rottlerin (data not shown).


Protein kinase C-δ inactivation inhibits the proliferation and survival of cancer stem cells in culture and in vivo.

Chen Z, Forman LW, Williams RM, Faller DV - BMC Cancer (2014)

Effects of a 3rd generation small molecule PKCδ inhibitor on human pancreatic cancer stem cell cultures. PCSC cells were grown to 80% confluence in 96-well plates and then exposed to BJE6-106 at concentrations ranging from 0.1 to 20 μM, or to rottlerin at concentrations ranging from 1 to 20 μM. The corresponding equivalent volume of solvent (DMSO) was used as a vehicle control (Vehicle). After 48 and 72 hr of exposure, cell mass was evaluated by MTT assay. Control values were normalized to 100%. Error bars represent SEM. p values for comparison between vehicle and rottlerin effects on cell number at 48 hr reached significance at 1 μM, and for BJE6-106 at 0.1 μM (p ≤ 0.02), and remained significant at the 72 hr time point.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3927586&req=5

Figure 3: Effects of a 3rd generation small molecule PKCδ inhibitor on human pancreatic cancer stem cell cultures. PCSC cells were grown to 80% confluence in 96-well plates and then exposed to BJE6-106 at concentrations ranging from 0.1 to 20 μM, or to rottlerin at concentrations ranging from 1 to 20 μM. The corresponding equivalent volume of solvent (DMSO) was used as a vehicle control (Vehicle). After 48 and 72 hr of exposure, cell mass was evaluated by MTT assay. Control values were normalized to 100%. Error bars represent SEM. p values for comparison between vehicle and rottlerin effects on cell number at 48 hr reached significance at 1 μM, and for BJE6-106 at 0.1 μM (p ≤ 0.02), and remained significant at the 72 hr time point.
Mentions: The activity of the 3rd generation PKCδ inhibitor BJE6-106 on the growth of PCSC cells in culture was compared to rottlerin. BJE6-106 inhibited the growth of PCSC cultures at concentrations as low as 0.1 μM, and had an (in culture) IC50 of approximately 0.5 μM at 48 hr (Figure 3). In contrast, rottlerin produced no significant inhibitory activity at 0.5 μM, and displayed an IC50 at 48 hr of approximately 3 μM. LDH release assays also showed greater than 10-fold increases in potency for BJE6-106 compared to rottlerin (data not shown).

Bottom Line: Inhibition of PKCδ by specific genetic strategies (shRNA) or by novel small molecule inhibitors is growth inhibitory and cytotoxic to multiple types of human CSCs in culture.PKCδ inhibition efficiently prevents tumor sphere outgrowth from tumor cell cultures, with exposure times as short as six hours.Small-molecule PKCδ inhibitors also inhibit human CSC growth in vivo in a mouse xenograft model.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Center, Boston University School of Medicine, K-712C, 72 E, Concord St,, Boston, MA 02118, USA. dfaller@bu.edu.

ABSTRACT

Background: A subpopulation of tumor cells with distinct stem-like properties (cancer stem-like cells, CSCs) may be responsible for tumor initiation, invasive growth, and possibly dissemination to distant organ sites. CSCs exhibit a spectrum of biological, biochemical, and molecular features that are consistent with a stem-like phenotype, including growth as non-adherent spheres (clonogenic potential), ability to form a new tumor in xenograft assays, unlimited self-renewal, and the capacity for multipotency and lineage-specific differentiation. PKCδ is a novel class serine/threonine kinase of the PKC family, and functions in a number of cellular activities including cell proliferation, survival or apoptosis. PKCδ has previously been validated as a synthetic lethal target in cancer cells of multiple types with aberrant activation of Ras signaling, using both genetic (shRNA and dominant-negative PKCδ mutants) and small molecule inhibitors. In contrast, PKCδ is not required for the proliferation or survival of normal cells, suggesting the potential tumor-specificity of a PKCδ-targeted approach.

Methods: shRNA knockdown was used validate PKCδ as a target in primary cancer stem cell lines and stem-like cells derived from human tumor cell lines, including breast, pancreatic, prostate and melanoma tumor cells. Novel and potent small molecule PKCδ inhibitors were employed in assays monitoring apoptosis, proliferation and clonogenic capacity of these cancer stem-like populations. Significant differences among data sets were determined using two-tailed Student's t tests or ANOVA.

Results: We demonstrate that CSC-like populations derived from multiple types of human primary tumors, from human cancer cell lines, and from transformed human cells, require PKCδ activity and are susceptible to agents which deplete PKCδ protein or activity. Inhibition of PKCδ by specific genetic strategies (shRNA) or by novel small molecule inhibitors is growth inhibitory and cytotoxic to multiple types of human CSCs in culture. PKCδ inhibition efficiently prevents tumor sphere outgrowth from tumor cell cultures, with exposure times as short as six hours. Small-molecule PKCδ inhibitors also inhibit human CSC growth in vivo in a mouse xenograft model.

Conclusions: These findings suggest that the novel PKC isozyme PKCδ may represent a new molecular target for cancer stem cell populations.

Show MeSH
Related in: MedlinePlus