Enhancing the Efficacy of HIPEC Through Bromelain: A Preclinical Investigation in Appendiceal Cancer

Abstract

Introduction: Appendiceal cancer (AC) excessive mucin production is a barrier to heated intraperitoneal chemotherapy (HIPEC) drug delivery. Bromelain is a pineapple stem extract with mucolytic properties. We explored bromelain treatment effects against mucinous AC in a patient-derived tumor organoid (PTO) model and an AC cell line.

Patients and methods: PTOs were fabricated from tumor specimens obtained from patients with AC undergoing cytoreductive surgery with HIPEC. PTOs underwent HIPEC treatment with bromelain, cisplatin, and mitomycin C (MMC) at 37 °C and 42 °C with and without bromelain pretreatment.

Results: From October 2020 to May 2023, 16 specimens were collected from 13 patients with low-grade (12/16, 75%) and high-grade AC (4/16, 25%). The mucin-depleting effects of bromelain were most significant in combination with N-acetylcysteine (NAC) compared with bromelain (47% versus 10%, p = 0.0009) or NAC alone (47% versus 12.8%, p = 0.0027). Bromelain demonstrated > 31% organoid viability reduction at 60 min (p < 0.001) and > 66% in 48 h (p < 0.0001). Pretreatment with bromelain increased cytotoxicity of both cisplatin and MMC HIPEC conditions by 31.6% (p = 0.0001) and 35.5% (p = 0.0001), respectively. Ki67, CK20, and MUC2 expression decreased after bromelain treatment; while increased caspase 3/7 activity and decreased Bcl-2 (p = 0.009) and Bcl-xL (p = 0.01) suggest induction of apoptosis pathways. Furthermore, autophagy proteins LC3A/B I (p < 0.03) and II (p < 0.031) were increased; while ATG7 (p < 0.01), ATG 12 (p < 0.04), and Becline 1(p < 0.03), expression decreased in bromelain-treated PTOs.

Conclusions: Bromelain demonstrates cytotoxicity and mucolytic activity against appendiceal cancer organoids. As a pretreatment agent, it potentiates the cytotoxicity of multiple HIPEC regimens, potentially mediated through programmed cell death and autophagy.

Keywords: Appendiceal cancer; Bromelain; HIPEC; Organoids.

Fig. 1

Schematic workflow of organoids fabrication from appendiceal tumor specimens and downstream analysis. Day 1 concludes with tumor cell isolation and encapsulation into the ECM-based hydrogel for organoid fabrication. After one week of culture, organoids are treated with HIPEC regimens for 72 hours, followed by analysis

Fig. 2

Mucolytic and cytotoxic effects of bromelain A Untreated mucin from ovarian metastasis from a low grade appendiceal cancer B Same mucin, 3 hours after treatment with 3% NAC, 600 mg/ml bromelain, bromelain and NAC (Brom + NAC), or PBS/untreated (control). C Pooled analysis of mucin degradation Plot (N=7) shows the percentage of residual mucin post-treatment under different conditions. P values of bromelain p < 0.0006, NAC p < 0.012, Brom+NAC p < 0.0001 compared to the control. D Pooled analysis (n=11) of ATP viability assay of organoids after treatment with 600 ug/ml bromelain for 60 min, 120 min, 48 h, and 72 h at 37 °C. (Mean ± SD, n = 11; **p < 0.01, ****p < 0.0001). AC PTO viability after 60 and 120 min of bromelain treatment was decreased by 31.2% and 32.2%, respectively. AC PTO Viability was further decreased by 65.9% after 48 hours and 74.8% after 72 h. E Corresponding Live/Dead stain at 48 and 72 hours showing > 43% and 80.2% decrease in viability, respectively. F show the AC PTO Immunohistochemical images of Ki67 (Alexa 488), ii. CK20 (Alexa 594), and iii. MUC2 (Alexa 594) following bromelain treatment. Nuclei were stained with DAPI

Fig. 3

Bromelain potentiates HIPEC effect when used as a pretreatment agent at 42 °C. A Pooled analysis (N=10) of ATP viability of AC PTOs after 1 hour of treatment with bromelain ± 1 additional hour of HIPEC with MMC, Cisplatin, and doxorubicin (42 °C). ATP viability data demonstrates synergistic action when PTOs are pre-treated with bromelain before HIPEC perfusates (Mean ± SD, N = 10; MMC vs Brom+MMC p < 0.0001, Cisplatin vs Brom+Cisplatin p < 0.0001, doxo vs Brom + doxo p < 0.02). PTOs 1-10 represent individual AC patients. B Representative Live/Dead stain imaged with confocal microscopy. Green color live cells. Red color dead cells, respectively. Scale bar 200 um

Fig. 4

The effect of bromelain on ATP viability of AC PTOs after 1 hour of treatment with bromelain followed by 1 hour of MMC or Cisplatin at 37 °C and 42 °C. PTOs are from 5 different specimens with PTOs 3 to 5 originating from spatially distinct sites of the same patient. Upper Row: Pooled (N = 5) ATP viability at 37 °C demonstrates synergistic action when PTOs are pre-treated with bromelain (Mean ± SD, MMC vs Brom+MMC p < 0.002, Cisplatin vs Brom+Cisplatin p < 0.03), that is less prominent when bromelain is heated at 42°C (MMC vs Brom+MMC p < 0.02, Cisplatin vs Brom+Cisplatin p < 0.06). Middle and lower row: ATP viability at 37 and 42 °C based on specimen

Fig. 5

Bromelain induces apoptosis in appendiceal PTOs observed through caspase activity and autophagy/apoptosis protein expression analysis. A Annexin V expression is in red, and caspase 3/7 activity expression is in green. Nuclei were stained with DAPI. B Pooled Caspase 3/7 activity from three individual patients. Bromelain-treated AC organoids show increased 3/7 activity compared to the control (p < 0.04, n = 3). Confocal images from A were quantitated in ImageJ. Bars represent the Mean fluorescence intensities (MFI) of Caspase 3/7 activity. C/F: Protein expression and densitometry shows trending inhibition of cyclin A2, Cyclin D1, Cyclin E1, and Cyclin H in bromelain-treated organoids (n=3). Densitometry of cell cycle protein expression normalized to GAPDH. D/H Densitometry of anti-apoptotic and pro-survival protein expression in Figure 5D. Figure 5H densitometry analysis shows significant inhibition of Bcl-2 p < 0.009 and Bcl-xL p < 0.01, n=3. E/G: represent the densitometry analysis of autophagy protein expression in Figure 5E. Densitometry analysis of autophagy protein expression shows that ATG7 p < 0.01, ATG 12 p < 0.04, and Beclin-1 expression p < 0.03 inhibit in bromelain-treated organoids. Expression of LC3 A/B I p <  0.01 and LC3 A/B II p < 0.03 both are increasing in bromelain-treated organoids compared to the control (n = 3)

Fig. 6

Effect of bromelain on Naxos 5 appendiceal tumor cell line organoids. A signet ring morphology on 10X H&E of Naxos 5 appendiceal cancer cell line organoids. B, C Confocal images of Naxos 5 organoids stained with live Mitoview and Caspase 3/7 substrate. Figure 6B represents organoids treated with bromelain for 48 hrs. Figure 6C control demonstrates untreated organoids. Red fluorescence in Figure 6B-C indicates intact mitochondrial membrane potential in organoids, and green fluorescence represents the caspase 3/7 activity. D Mean fluorescence intensity (MFI) of red fluorescence representing intact mitochondrial membrane potential (B, C) in untreated vs bromelain-treated organoids. Bromelain treatment significantly inhibits mitochondrial membrane potential (87.87% compared to the control) with p < 0.02 control vs bromelain (n = 3). E: Pooled ATP viability of Naxos 5 organoids after 1 hour of treatment with bromelain ± 1 additional hour at 37 °C with MMC and Cisplatin. ATP viability data reveals synergistic action when organoids are pre-treated with bromelain at 37 °C (Mean ± SD, n = 3; MMC vs Brom + MMC p < 0.007, Cisplatin vs Brom + Cisplatin p < 0.003. F HIPEC 42 °C Pooled (N = 3) ATP viability of Naxos 5 organoids after 1 hour of treatment with bromelain ± 1 additional hour of HIPEC with heated MMC, and Cisplatin (42 °C). MMC vs Brom + MMC p < 0.0001, Cisplatin vs Brom+Cisplatin p < 0.006