Neural stem/progenitors and glioma stem-like cells have differential sensitivity to chemotherapy

Abstract

Objectives: New data suggest that glioma stem-like cells (GSCs) and neural stem/progenitor cells (NSCs) may share common origins. GSCs drive tumor proliferation and appear to be resistant to classic chemotherapy, while the effects of chemotherapy on NSCs are not well studied. As the role of NSCs in learning and memory is increasingly recognized, we need to identify drugs that reduce neurotoxicity but are still effective against glial tumors.

Methods: We treated 3 human NSC cultures and multiple low- and high-grade GSC cultures with the commonly used agents temozolomide (TMZ) and cisplatin (CIS), and with 2 newer, promising drugs: the proteasome inhibitor bortezomib (BTZ) and the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib (ERL). We measured cell survival, proliferation, cell death induction, and drug resistance markers.

Results: TMZ decreased NSC viability, while minimally affecting GSCs. TMZ induced NSC death, which was partially compensated for by increased proliferation. CIS had similar effects. The NSC's sensitivity to TMZ and CIS correlated with low expression of the multidrug resistance gene ABCG2, but not of MGMT or MSH1/MLH2. BTZ caused an 80%decrease in GSCs, while minimally affecting NSCs. GSCs had lower proteasome levels and activity after BTZ treatment. ERL treatment also decreased GSC numbers, but not NSC viability, which correlated with low EGFR expression in NSCs compared to GSCs.

Conclusions: Newer chemotherapy agents ERL and BTZ are effective against GSCs yet produce minimal effects on NSCs, while the older drugs TMZ and CIS are more toxic for NSCs than for GSCs. The identification and testing of more selective drugs is clearly warranted.

Figure 1. Human neural stem/progenitor cells (NSCs) are more sensitive to temozolomide (TMZ) than glioma stem-like cells (GSCs)

(A) In vitro treatment with graded doses of TMZ leads to a decreased number of NSCs (SC27) while minimally affecting GSCs (HuTuP01). (B) Treatment with 200 μM of TMZ kills 50% of the neural stem cells (SC23, SC27, and SC30). The same dose has less toxicity for low-grade (DB01, DB06) and high-grade (DB05, DB17, HuTu) GSCs. (C) Cellular proliferation of NSC as measured by BrdU incorporation is up to 8 times higher 3 days after the treatment with low doses of TMZ, but returns to lower levels 7 days after treatment (D). Seven days after TMZ treatment, no significant effects on proliferation are seen in GSCs.

Figure 2. Temozolomide (TMZ) induces cell death in neural stem/progenitor cells (NSCs) but not in glioma stem-like cells (GSCs) by a methyl-guanine methyl transferase (MGMT)–independent mechanism

(A) Seven days after TMZ treatment, 25% of NSCs (SC23) treated with 500 μM TMZ and 17% of NSCs treated with 200 μM TMZ were positive for PI staining by flow cytometry, consistent with increased membrane permeability indicative of necrotic cell death, while GSCs (DB 17) were not affected. (B) NSCs and GSCs have similar gene expressions for the enzyme controlling resistance to TMZ (MGMT), but much higher expressions than the stable glioma cell lines. The data are average values of 3 NSC cultures, 11 GSC cultures, and the 2 stable glioma lines.

Figure 3. Neural stem/progenitor cells (NSCs) are more sensitive to cisplatin (CIS) than glioma stem-like cells (GSCs) and display lower expression levels for ABCG2 multidrug resistance gene, but similar levels for the mismatch repair enzyme genes

(A) In vitro treatment with graded doses of CIS leads to a decreased number of NSCs (SC23, SC27) while minimally affecting GSCs (HuTu, DB17). (B) Cellular proliferation of NSCs, as measured by BrdU incorporation, is up to 10 times higher 3 days after the treatment with low doses of CIS, but returns to lower levels 7 days after treatment (data not shown). (C) NSCs and GSCs have similar gene expressions for the mismatch repair enzymes controlling resistance to CIS (MSH1, MLH2). (D) NSCs have significantly lower expression of the ABCG2 multidrug resistance gene. The data are average values of 3 NSC cultures, 11 GSC cultures, and the 2 stable glioma lines.

Figure 4. Neural stem/progenitor cells (NSCs) are less sensitive to proteasome inhibition than glioma stem-like cells (GSCs) due to higher proteasome expression and function and lower caspase-3 activation

(A) In vitro treatment with graded doses of bortezomib (BTZ) kills off the GSCs (HuTuP01) while minimally affecting NSCs (SC27). (B) Treatment with 5 nM of BTZ killed 75%–90% of the low-grade (DB01, DB06) and high-grade (DB05, DB07, DB17, HuTu) GSCs. The same dose had less toxicity for NSCs (SC27 and SC30). (C) Caspase-3 activity was measured in cell extracts, 24 hours after treatment with BTZ (5 nM) and ZVAD (50 μM), as indicated. GSCs had high levels of caspase-3 activation after the BTZ treatment, which was reversed by ZVAD addition, while the caspase-3 levels in NSC did not increase after BTZ treatment. (D) Proteasome activity was measured in cell extracts, at baseline and 4 hours after treatment with BTZ (5 nM). NSCs had higher baseline proteasome levels than GSCs, which remained higher than the GSC levels after the proteasome inhibition. (E, F) NSCs had significantly higher expression of the proteasome subunits PSMB5, PSMB6, and PSMB7. The data are average values of 3 NSC cultures, 11 GSC cultures, and the 2 stable glioma lines.

Figure 5. EGFR pathway inhibition with ERL preferentially affects glioma stem-like cells (GSCs) and correlates with EGFR expression

(A) In vitro treatment with graded doses of ERL led to a decreased number of GSCs (HuTu, DB17) while minimally affecting neural stem/progenitor cells (NSCs) (SC23, SC27). (B.a) GSCs are overwhelmingly positive for EGFR staining while (B.b) NSCs have low and sporadic expression (3 NSCs cultures and 5 GSCs cultures tested, representative results shown).