>>7435118

Original Study

DEC 2019

The phenanthrene derivative PJ34 exclusively eradicates human pancreatic cancer cells in xenografts

http://www.oncotarget.com/index.php?journal=oncotarget&page=article&op=view&path[0]=27268&path[1]=87898

http://archive.is/EkHBB

ABSTRACT

Recent reports demonstrate an exclusive eradication of a variety of human cancer cells by the modified phenanthridine PJ34. Their eradication during mitosis is attributed to PJ34 preventing NuMA clustering in the mitotic spindle poles of human malignant cells, which is crucial for their normal mitosis. Here, the effect of PJ34 is tested in cell cultures and xenografts of human pancreas ductal adenocarcinoma. Evidence is presented for a substantial reduction (80–90%) of PANC1 cancer cells in xenografts, measured 30 days after the treatment with PJ34 has been terminated. Benign cells infiltrated into the PANC1 tumors (stroma) were not affected. Growth, weight gain and behavior of the treated nude mice were not impaired during, and 30 days after the treatment with PJ34. The efficient eradication of malignant cells in human pancreas cancer xenografts presents a new model of pancreas cancer treatment.

Introduction'

Despite a substantial advance in cancer treatment, pancreatic ductal adenocarcinoma (PDAC) have a limited response to current treatments, and a low 5-years survival rate of about 6% [1–3]. Thus, there is an urgent need to explore new mechanisms for treating this lethal malignancy.

Recent reports have discovered the capability of phenanthrenes to kill human cancer cells that are resistant to currently prescribed apoptosis-inducing agents [4, 5]. Furthermore, we identified phenanthrenes acting as PARP1 inhibitors that efficiently eradicate a variety of human cancer cells without impairing benign cells [6–9]. Notably, their exclusive cytotoxic activity in human cancer cells was independent of, and un-related to PARP1 inhibition [7–11]. The phenanthrenes PJ34, TiqA and phenanthridinon (Phen) act as PARP1 inhibitors due to their binding potency to the nicotine-amide binding site in the catalytic domain of PARP1 [12, 13]. However, their PARP1 inhibition per-se does not impair nor eradicate human malignant cells, including pancreas cancer cells, PANC1 [7–9]. In contrast, at higher concentrations than those causing PARP1 inhibition, PJ34, Tiq-A and Phen eradicate a variety of human cancer cells by ‘mitotic catastrophe cell death’. This cell-death follows mitosis arrest caused by preventing the post translational modification of NuMA (Nuclear mitotic apparatus protein-1) that enables its binding to proteins [8].

In the tested human cancer cells, NuMA binding to proteins enables its clustering in the spindle poles, which is crucial for stabilizing the spindle, a pre-requisite for chromosomes alignment in the spindle mid-zone and normal anaphase. Notably, NuMA silencing or down regulation of NuMA prevents mitosis in all cell types [14–17].

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cont'd

In human malignant cells, specific post translational modifications of NuMA enable and promote NuMA binding to proteins [18–21]. These post translational modifications are most efficiently prevented by PJ34 [8, 20, 21]. In accordance, in PJ34 treated cancer cells, NuMA is arbitrarily dispersed in the spindle, instead of being clustered in the spindle poles [8]. The consequences are un-stabilized spindle pole with dispersed chromosomes, instead of segregated chromosomes aligned in the spindle mid-zone [8, 17, 22, 23]. This abnormality jeopardizes normal ploidy of the ‘daughter’ cells and evokes mitosis arrest [17, 23]. Cells with these abnormal spindles are eradicated by a rapid death mechanism, ‘mitotic catastrophe’ cell-death [9, 23].

Here, the efficacy of PJ34 to eradicate human pancreas cancer cells is tested in cell cultures and in xenografts. PANC1 cells are most frequently identified in human pancreas tumors [1–3]. Pancreas xenografts were developed in nude mice. These tumors also contained normal infiltrating cells (stroma) of mouse origin [24–27], i. e. fibroblasts, myofibroblasts, macrophages and lymphocytes infiltrated into the tumors [25, 26].

Mitosis arrest and cell death were measured in PANC1 cells incubated with PJ34. In xenografts, eradication of human PANC1 cells deduced from a massive reduction of human proteins in the tumors, was measured 30 days after the treatment with PJ34 has been terminated. An increased necrosis measured in the PANC1 tumors of mice treated with PJ34 supports cell-death caused by PJ34 in the xenografts. Normal cells infiltrated into the tumors were not impaired by PJ34. A similar cytotoxic activity of PJ34 was observed in patients-derived PDAC cells and xenografts. These results indicate the potency of PJ34 to eradicate human pancreas cancers.

Results

Treatment with PJ34 causes mitosis arrest and cell death in human pancreas cancer cells PANC1

Measuring changes in the ploidy of PJ34 treated PANC1 cells with stained DNA by flow cytometry reveals piled up PANC1 cells with double DNA content, unable to proceed to mitosis (Figure 1). A similar cytotoxic effect of PJ34 is measured in other human malignant cell types [7]. Recently, the molecular mechanism causing mitosis failure and arrest in human cancer cells incubated with PJ34 has been disclosed [8]. The cell cycle profile of PANC1 cells incubated with PJ34 reflects their mitosis arrest and cell death (Figure 1). PJ34 (20 µM or 30 µM) was applied 24 hour after seeding, and PANC1 cell eradication and the kinetics of S-phase entry and G2/M transition were measured by flow cytometry after 48, 72 and 120 hours incubation (Methods). After 48 hours incubation with PJ34, failure to proceed into mitosis preceded cell death measured after 72 hours incubation. These cells were eradicated after 120 hour incubation with PJ34 (Figure 1).