Primary and liver metastasis-derived cell lines from KrasG12D; Trp53R172H; Pdx-1 Cre animals undergo apoptosis in response to triptolide

Abstract

Objectives: Pancreatic cancer has a 5-year survival rate of less than 5%, partly because of limited chemotherapeutic options, thereby highlighting the need for novel therapies. Triptolide, a diterpene triepoxide that was derived from a Chinese herb, has shown great promise in preclinical testing against pancreatic cancer using immunocompromised animals.

Results: In this study, we tested the ability of triptolide to induce cell death in cell lines derived from a primary tumor and adjacent liver metastases of immunocompetent animals (Kras, Trp53, Pdx-1 Cre [KPC]). Both cell lines were more aggressive in their ability to form tumors when compared with other pancreatic cancer cell lines and showed constitutive activation of the nuclear factor κ-light-chain-enhancer of activated B cells pathway. Triptolide induced apoptotic cell death in both cell lines, as evidenced by decreased cell viability as well as increased caspase 3/7 activity, annexin V positivity, and increased terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positivity in tumors from KPC animals treated with Minnelide. In addition, triptolide decreased levels of HSP70, its transcription factor HSF1, as well as the antiapoptotic proteins Bcl-xL, Bcl-2, and Mcl-1, which are known to be up-regulated in pancreatic cancer.

Conclusions: The ability of triptolide to cause cell death in cell lines derived from immunocompetent animals further validates its potential as a novel agent against pancreatic cancer.

Figure 1. Cell lines derived from both primary and liver metastasis form tumors in syngenic animals

A. Pictorial representation of animal used to obtain tissue from which both the KPC1 and KPC1-LM cell lines were derived. B. KPC1 (top panel) and KPC1-LM (bottom panel) cells are able to form tumors in syngenic immune-competent animals. 5 × 10⁴ cells were injected into the flank of C57 BLK6 animals and animals sacrificed 28 days post-injection.

Figure 2. Tumors derived from KPC1 and KPC1-LM cells show greater tumorigenicity compared to other pancreatic cancer cell lines

A. Cells were injected into flanks of athymic nude animals and their ability to form tumors assessed. Animals were sacrificed 21 days after cell injection. B. Tumor volume, and C. Tumor weight of tumors derived from pancreatic cancer cells (KPC1, KPC1-LM, KPC023, MIA PaCa-2, AsPC-1, S2-013 and S2-VP10). Errors bars represent SEM. *= p ≤ 0.05 vs. KPC1

Figure 3. NF-κB is constitutively active in pancreatic cancer cell-derived tumors

Protein lysates from tumors of animals injected with pancreatic cancer cell lines indicated were assessed for the activity of the p50 subunit of NFkB and normalized to protein concentration. *= p ≤ 0.05 vs. KPC1

Figure 4. Triptolide causes cell death in KPC tumor-derived cells

Cells were treated with triptolide (0–200 nM) and viability assessed at 6, 12, 24, 48 and 72h. Both KPC1 and KPC1-LM cells show decreased cell viability in response to triptolide. *= p ≤ 0.05 vs. untreated controls.

Figure 5. KPC tumor derived cell lines undergo apoptotic cell death in response to triptolide

A–D. Cells were treated with triptolide (0–200 nM) and caspase 3/7 activity and number of Annexin positive cells assessed at times indicated. A. and B. Activation of caspase 3/7 was assessed using Caspase-Glo and normalized to viability. C and D. Annexin positivity was assessed using flow cytometry. Data is represented as percent untreated cells. Both KPC1 and KPC1-LM cells show an increase in caspase activiation and Annexin positivity. Errors bars represent SEM. E. KPC animals were injected with either saline or Minnelide, the water soluble prodrug of triptolide for 7 days and tumors harvested. TUNEL staining on formalin fixed sections of the tumors show greater positivity in response to Minnelide treatment. *= p ≤ 0.05 vs. untreated control.

Figure 6. Anti-apoptotic proteins are down regulated in response to triptolide in KPC and KPC1-LM cells

A. Cells were treated for 24 hours with 100 nM triptolide and gene expression evaluated using real time PCR. 18s was used to normalize expression and data represented as fold change compared to control untreated cells. Errors bars represent SEM. *= p ≤ 0.05 vs. untreated cells. B. Protein lysates from cells treated with triptolide as described in A. were immuno-blotted for HSP70 or HSF1. C. Protein lysates from cells treated with triptolide as described in A. were immuno-blotted for Bcl-2, Bcl-xl and Mcl-1. Actin was used as a loading control for all western blots.