Volume 71, Issue 2 p. 147-154
Original Article
Free Access

Effects of anaesthesia on proliferation, invasion and apoptosis of LoVo colon cancer cells in vitro

Y. J. Xu

Y. J. Xu

Associate Professor

Department of Anaesthesiology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China

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S. Y. Li

S. Y. Li

Associate Professor

Pathology Office, Shanghai University of Tranditional Chinese Medicine, Shanghai, China

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Q. Cheng

Q. Cheng

Resident

Department of Anaesthesiology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China

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W. K. Chen

W. K. Chen

Attending Doctor

Department of Anaesthesiology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China

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S. L. Wang

S. L. Wang

Attending Doctor

Department of Anaesthesiology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China

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Y. Ren

Y. Ren

Attending Doctor

Department of Anaesthesiology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China

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C. H. Miao

Corresponding Author

C. H. Miao

Professor and Chief

Department of Anaesthesiology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China

Correspondence to: C. Miao

Email: [email protected]

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First published: 16 December 2015
Citations: 54

This article is accompanied by an editorial by Ciechanowicz and Ma, Anaesthesia 2016; 71: 127–31.

You can respond to this article at http://www.anaesthesiacorrespondence.com

Summary

Tumour cell proliferation, invasion and apoptosis are crucial steps in tumour metastasis. We evaluated the effect of serum from patients undergoing colon cancer surgery receiving thoracic epidural and propofol anaesthesia on colon cancer cell biology. Patients were randomly assigned to receive propofol anaesthesia with a concomitant thoracic epidural (PEA, n = 20) or sevoflurane anaesthesia with opioid analgesia (SGA, n = 20). Venous blood was obtained before induction of anaesthesia and 24 hours postoperatively. The LoVo colon cancer cells were cultured with patient serum from both groups and the effects on proliferation, invasion and apoptosis were measured. Twenty-four hours after surgery, the absorbance value of LoVo cells at 10% serum concentration from PEA was decreased when compared with SGA (0.302 (0.026) vs 0.391 (0.066), p = 0.005). The inhibitory rate of LoVo cells at 10% serum concentration from PEA was higher than that from SGA (p = 0.004) 24 h after surgery. The number of invasive LoVo cells at 10% serum concentration from PEA was reduced when compared with SGA (44 (4) vs 62 (4), p < 0.001). Exposure of LoVo cells to postoperative serum from patients receiving PEA led to a higher luminescence ratio (apoptosis) than those receiving SGA (0.36 (0.04) vs 0.27 (0.05), p < 0.001). Serum from patients receiving PEA for colon cancer surgery inhibited proliferation and invasion of LoVo cells and induced apoptosis in vitro more than that from patients receiving SGA. Anaesthetic technique might influence the serum milieu in a way that affects cancer cell biology and, thereby, tumour metastastasis.

Introduction

Surgery is the primary treatment for colon cancer. However, the rate of recurrence or metastasis in colon cancer can be as high as 30%, even in stages 1 and 2 1. Most colon cancer-related deaths are caused by metastatic disease 2, 3. Tumour metastasis is a complicated process. The metastatic process includes: cellular invasion of and migration through surrounding tissues; invasion of the intravascular space; and proliferation in a target organ 4, 5. Apoptosis acts as a critical regulator in cancer cell growth, including metastasis and recurrence. Multiple factors, including immune cytokine signalling, can affect apoptosis 6, 7.

During the peri-operative period, the minimal residual cancer (MRC) includes micrometastatic deposits, scattered tumour cells during surgery, and circulating cancer cells. Peri-operative immunosuppression, the surgical stress response, pain and opioids could plausibly affect this proliferation, invasion and apoptosis in MRC. It has been suggested that anaesthetic technique can potentially modulate these factors 8, 9.

Inflammation plays a central role in cancer. To assess inflammation, the neutrophil/lymphocyte ratio (NLR) can act as a prognostic factor in surgical colon cancer 10, 11. The effect of anaesthetic technique on NLR is unknown. Thoracic epidural anaesthesia (TEA) can increase intestinal perfusion, attenuate leucocyte adhesion, and decrease patient morbidity in systemic inflammation by blocking regional sympathetic nervous system activity 12-14. Recently, we have found that thoracic epidural anaesthesia decreases serum levels of angiogenesis factors in open colon cancer surgery 15. Although previous studies suggested that serum from patients receiving propofol anaesthesia with paravertebral block for breast cancer surgery inhibited proliferation and induced apoptosis in vitro 16, 17, the effect of thoracic epidural anaesthesia on cancer cell biology is unknown.

We took serum from patients undergoing open colon cancer surgery with two different anaesthetic techniques, and evaluated its effects on proliferation, invasion and apoptosis of the human colon cancer cell line LoVo in vitro. The LoVo cell line was derived from a metastatic tumour in the left supraclavicular region of a 56 year-old male with adenocarcinoma of the colon.

Methods

With approval from the Cancer Hospital, Fudan University Institutional Human Ethics Committee (Shanghai, China) and written informed consent, patients aged 30–81 years undergoing open colon cancer surgery were enrolled in this randomised trial. The exclusion criteria were patients with general contraindications to epidural anaesthesia a history of chemotherapy or radiation within eight weeks or any contra-indication to the use of midazolam, sufentanil, propofol or sevoflurane. According to computer-generated codes, patients were randomly allocated to receive either propofol anaesthesia with a thoracic epidural for analgesia (PEA), or sevoflurane anaesthesia with opioid analgesia (SGA). In the PEA group, a catheter was inserted in the T9-12 interspace and advanced four cm in a cephalad direction. The test dose with lignocaine 1% was 3 ml. A bolus dose of 6–8 ml ropivacaine 0.375% was injected at least 20 min before surgery. Ropivacaine 0.375% was then infused at a rate of 5 ml.h−1 for the duration of surgery. General anaesthesia was induced with midazolam 0.03 mg.kg−1, sufentanil 0.3 μg.kg−1 and a propofol target-controlled infusion (TCI 3.5–4 μg.ml−1 using the Marsh pharmacokinetic model). Cisatracurium 0.2 mg.kg−1 was administered before tracheal intubation. Anaesthesia was maintained with propofol TCI (a mean plasma concentration of 2.6–3.2 μg.ml−1). The epidural regimen consisted of ropivacaine 0.15% and sufentanil 0.5 μg.ml−1 with a background infusion rate of 4 ml.h−1, a 3 ml bolus by patient control and a 15 min lockout time (PCEA).

Induction of anaesthesia for patients in the SGA group consisted of midazolam 0.03 mg.kg−1, sufentanil 0.3 μg.kg−1, and propofol 1–2 mg.kg−1. Cisatracurium 0.2 mg.kg−1 was administered before tracheal intubation. Anaesthesia was maintained using sevoflurane (at 1.0–1.5 minimum alveolar concentration). Intra-operative analgesia consisted of sufentanil 0.2–0.4 μg.kg−1.h−1. Patient-controlled intravenous analgesia (PCIA) was used postoperatively with sufentanil (1 μg.ml−1). The PCIA was set at a bolus of 2 ml, a 15-min lockout time, and a background infusion rate of 2.5 ml.h−1. In both groups, room temperature was maintained at 22 °C–25 °C and oesophageal temperature above 36 °C during surgery with a forced-air warming blanket (Inditherm Patient Warming System, Rotherham, UK). During surgery the bispectral index (BIS; Aspect Medical Systems, Newton, MA, USA) level was maintained between 45 and 55 in both groups. The analgesia was discontinued on day three. The visual analogue scale (VAS) was used to assess pain scores at rest and during coughing at two h, 24 h and 48 h postoperatively. Venous blood was obtained before induction and 24 h postoperatively. Samples were centrifuged at 4000 rpm. Thereafter, the serum was stored at −80 °C for future use.

The human colon cancer LoVo cell line was purchased from the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% foetal bovine serum (FBS), 100 units.ml−1 of penicillin and 100 mg.ml−1 of streptomycin at 37 °C in a humidified 5% CO2 atmosphere. Cell proliferation was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. LoVo cells were seeded in 96-well plates (1 × 104 cells per well). To allow cell attachment, the culture plates were cultured in a serum-free medium (not containing patient serum) for 24 h at 37 °C. Serum samples were diluted in medium to make 2%, 5% and 10% concentrations. These concentrations were in accordance with standard practice in tissue culture experiments in vitro. Medium alone acted as a control. Subsequently, 20 μl of MTT reagent (Sigma, St. Louis, MO, USA; 5 mg. ml−1) was added to the medium. The reaction was allowed to proceed for four h at 37 °C before dimethyl sulphoxide (DMSO; 150 μl) was added to each well to quench the reaction, with the plate shaken on a rotary platform for ten min. The absorbance was measured at 490 nm. The inhibition rates of proliferation in LoVo cells were calculated according to the following formula: % proliferation inhibition = (1 − OD490 of test well)/(OD490 of control well) × 100%.

The in vitro invasive ability of LoVo cells was tested using the Boyden chamber invasion assay. Polycarbonate membranes with a pore size diameter of 8.0 μm were spread with 50 μl (10 μg) of Matrigel (BD Biosciences, Bedford, MA, USA) on the upper surface of transwell cell culture chambers (Costar 3422, Cambridge, MA, USA) and incubated at 37 °C. The cells were trypsinised and incubated to the upper part of the Boyden chamber at a density of 3 × 105 cells.ml−1 in 300 μl of serum-free medium. The bottom chamber contained medium with 10% FBS as a chemo-attractant. Patient serum samples were diluted with medium to a 10% concentration. A total of 100 μl of both pre- and postoperative serum samples from both the PEA and SGA groups were added in duplicate to both upper and lower chambers of the appropriate wells. LoVo cells were then suspended in 100 μl RPMI 1640 containing FBS 0.1%, added to the upper compartment and cultured at 37 °C for 24 h. The filters were fixed with 95% ethanol for 15 min and stained with crystal violet 0.05% in distilled water. After gently rinsing with water, the cells on the upper surface of the filters were removed by wiping with a cotton swab. The stained cells invading to the lower surface were photographed. The number of cells penetrating the membrane was counted using an optical microscope (×200 magnification).

Cell viability and apoptosis were evaluated using ApoLive-Glo Multiplex Assay from Promega (Southampton, UK). LoVo Cells were cultured in L-15 Medium (Leibovitz) containing 10% FBS, 1% penicillin-streptomycin solution and L-glutamine at 37 °C with CO2 5% for 48 h. The cells were then harvested by trypsinisation, resuspended in medium, and added to clear-bottom 96-well plates at a density of 5000 cells.ml−1. To allow cell attachment, culture plates were subsequently cultured in full medium for 24 h at 37 °C. Serum was diluted in medium to produce 10% serum concentrations, which was added in triplicate. Culture plates were incubated for a further 24 h. 20 μl Viability Reagent was then added to the wells and mixed by orbital shaking. Plates were cultured for 30 min at 37 °C. Fluorescence was analysed at 400Ex/505Em nm (viability). A total of 100 μl Caspase-Glo 3/7 reagent was added and mixed briefly by orbital shaking (300–500 rpm for 30 s). Plates were then left for 30 min at room temperature. GloMax-Multi Microplate Multimode Reader (Glomax, Promega, Southampton, UK) was used to measure luminescence.

The SPSS 16.0 software package (SPSS Inc., Chicago, IL, USA) was used for analysis. According to previously published studies of breast cancer cells in vitro 16, 17, we did not prospectively calculate a sample size to detect a specific change in colon cancer cell function. Continuous data was compared using an independent group t-test. Differences between different serum concentrations were assessed using repeated-measures analysis of variance and corrected with a Tukey's post-hoc test. Categorical variables such as patient sex, ASA physical status, patients with anaemia or diabetes mellitus, patients receiving blood transfusion and tumour stage were analysed using Pearson chi-square test or Fisher's exact test. Luminescence data were normalised to control luminescence without serum and expressed as a ratio of control luminescence.

Results

All patients were studied. The same surgical and anaesthetic team performed all procedures. The two groups were comparable in terms of patient characteristics and clinical data (Table 1). The use of sufentanil was significantly lower in the PEA group compared with the SGA group (20 (3.2) vs 51 (5.5) μg, p < 0.001) (Table 1).

Table 1. Baseline characteristics and clinical data. Values are mean (SD) or number (proportion)
General anaesthesia (n = 20) Thoracic epidural (n = 20) p value
Age; years 59 (10) 55 (12)
Sex; M/F 12/8 13/7
Weight; kg 65 (8) 64 (12)
Height; cm 164 (6) 167 (8)
ASA I/II/III 2/16/2 4/14/2
Anaemia 4 (20%) 5 (25%)
Albumin; g.l−1 41 (2.8) 40 (3.0)
Diabetes mellitus 5 (25%) 4 (20%)
Blood transfusion 4 (20%) 5 (25%) 0.70
Blood loss; ml 240 (104) 250 (115) 0.80
Time of surgery; min 118 (38) 102 (29) 0.78
Tumour stage
I 5 3 0.73
II 7 8
III 8 9
Sufentanil (μg) 51 (5.5) 20 (3.2) < 0.001

Before surgery, the two groups did not differ significantly in cellular proliferation after treatment of cells with 0%, 2%, 5% and 10% patient serum (Fig. 1a). Twenty-four h after surgery, however, the absorbance value of LoVo cells at 10% serum concentration from PEA was significantly reduced compared with SGA (0.302 (0.026) vs 0.391 (0.066), p = 0.005) (Fig. 1b). The inhibition rate of LoVo cells at 10% serum concentration from PEA was significantly higher than that from SGA (23.2% vs 42.2%, p = 0.004). The number of invasive LoVo cells at 10% serum concentration from PEA was similar compared with SGA (33 (4) vs 35 (5), p = 0.141) before surgery (Fig. 2). The number of invasive LoVo cells at 10% serum concentration from PEA was significantly reduced compared with SGA (44 (4) vs 62 (4), p < 0.001) 24 h after surgery (Fig. 3).

Details are in the caption following the image
Effect of 0%, 2%, 5% and 10% serum on LoVo cell proliferation. White, general (SGA); black, epidural (PEA) group. Data are mean (SD). *p = 0.005 vs epidural group.
Details are in the caption following the image
Effect of 10% serum on invasion on LoVo cell (pre). Data are mean (SD).
Details are in the caption following the image
Effect of 10% serum on invasion on LoVo cell (post 24 h). Data are mean (SD). *p < 0.001 vs epidural group.

Exposure of LoVo cells to the postoperative serum of SGA patients led to a higher cell viability ratio than PEA patients (0.54 (0.08) vs 0.48 (0.06), P = 0.01) (Fig. 4a). Exposure of LoVo cells to the postoperative serum of PEA patients led to higher luminescence ratio (apoptosis) than SGA patients (0.36 (0.04) vs 0.27 (0.05), p < 0.001) (Fig. 4b). The luminescence ratio (apoptosis) of postoperative serum from SGA was reduced compared with pre-operative SGA (0.27 (0.05) vs 0.32 (0.05), p = 0.002) (Fig. 4b).

Details are in the caption following the image
Effect of 10% serum on LoVo cell viability (a) and apoptosis (b). Data are mean (SD). White, general (SGA); black, epidural (PEA) group. *p = 0.01 vs epidural group; #p < 0.001 vs sevoflurane group; p = 0.002 vs pre-operative.

The two groups were similar in NLR before surgery. The NLR was lower in patients receiving PEA at 24 h (9.15 (0.81) vs 12.1 (1.61), p = 0.001) and 72 h (5.1 (0.76) vs 8.6 (1.53), p = 0.029) (Fig. 5) after surgery, compared with patients receiving SGA.

Details are in the caption following the image
Time course of neutrophil:lymphocyte ratio. Data are mean (SD). White, general (SGA); black, epidural (PEA) group. *p < 0.05 vs epidural group.

Discussion

The primary cause of morbidity and mortality in colon cancer is the ability of cancer cells to metastasise to different sites in the body. The metastatic process includes several steps. The steps consist of growth of the tumour at the primary site; angiogenesis; invasion of this new vasculature by cancer cells; and extravasation at distant sites. Surgery itself can contribute to metastasis as tumour cells can be unintentionally disseminated into the circulation 4, 18, 19. Surgical resection of the tumour may also lead to an increase in angiogenic and growth factors, both of which have tumour-promoting effects 20. Surgery also induces a ‘stress’ response with neuro-endocrine, cytokine and metabolic components 21, 22. This complex interplay of factors can affect the tumour micro-environment during the peri-operative period such that pre-existing quiescent micrometastasis or surgically disseminated cancer cells may become active. Recent data suggest that choice of anaesthetic technique may affect cancer outcome. Most research has focused on the interplay between local anaesthetics/regional anaesthesia and cancer recurrence 8, 9, 23-25 compared with opioid use.

Cell mediated immunity (CMI) plays an important role in cancer development 26 and anaesthetics can affect this process via multiple interactions 27. Regional anaesthesia provides excellent pain relief and reduces neuroendocrine and metabolic stress and inflammatory response as well as reducing anaesthesia and opioid requirements.

We chose the colon LoVo cell line as it is highly aggressive both in vitro and in vivo, and associated clinically with a worse overall prognosis 28, 29. Therefore, if anaesthesia administered for colon cancer surgery could attenuate the proliferation and invasion or increase apoptosis of this highly aggressive cell line, this could have strong clinical significance. Apoptosis is the process of programmed cell death. It can be deviant and lead to cancer metastasis. Proteases are known as caspases and central to apoptotic mechanisms. Caspase-3 activation is known as a marker for apoptosis. In our study, the Glomax assay used caspase-3 activation, but we do not know whether the effects we observed were mediated by a caspase-3 mechanism. A causative factor for development and promotion of cancer is defective apoptosis 30. Volatile anaesthetics may influence apoptotic mechanisms in different cell types. Previous work has shown that volatile anaesthetics may protect against apoptotic cell death in normal myocardial cells 31. Isoflurane attenuates the apoptotic effect of tumour necrosis factor through a mechanism involving caveolin-1 in human colon cancer cells 32. Recently, two studies showed that lignocaine and bupivacaine induce apoptosis in breast and thyroid cancer cells 33, 34. Many patients with colon cancer harbour micrometastases and disseminated tumour cells at the time of surgery 1, 4. Whether the micrometastases develop into clinically significant metastases depends on the immune system's ability to eradicate them. Our study demonstrated that cellular proliferation and invasion of the human colonLoVo cell was significantly reduced when cells were treated with serum from the PEA group at 10% serum concentration. In contrast, apoptosis of colon LoVo cells was significantly reduced when they were treated with serum from the SGA group at 10% concentration. Volatile anaesthetics and propofol appear to have different effects on cancer cell biology. Isoflurane, but not propofol can increase tumour growth in a human cancer cell line, whereas propofol may kill cancer cells 35-37. There is also data showing that local anaesthetics may directly kill cancer cells 38. Recently, our group found that the use of PEA compared with SGA alone for colon cancer surgery led to a reduction in serum expression of angiogenesis-promoting factors 15, so a possible explanation of our finding is that the molecular profile of the serum of patients with colon cancer may be changed by the anaesthetic technique. Many clinical studies, however, are small or retrospective in nature, and prospective, large-scale clinical trials will be required to determine whether anaesthetic technique can conclusively affect cancer outcome.

The neutrophil/lymphocyte ratio is a simple parameter which is associated with systemic inflammation, and can be used as a prognostic factor in patients with cancer having surgery 10, 39. Tumo-ur-associated neutrophils are involved with the production of prostaglandin-E2 (PGE2), reactive oxygen species, basic fibroblast growth factor and vascular endothelial growth factor, which all lead to more aggressive tumour behaviour 40. In our study, we found that NLR was lower in patients receiving PEA at 24 h and 72 h after surgery.

There are three main limitations to our study. First, the sample size is relatively small. As this was performed as a pilot research study, we did not prospectively calculate a sample size to detect a specific change in colon cancer cell proliferation, invasion and apoptosis. Second, the serum levels of ropivacaine were not examined, so it was difficult to determine whether the change in colon cell function was influenced directly by ropivacaine in the serum or blockade of the sympathetic nervous system. Third, PEA patients received both epidural and propofol-based anaesthesia as opposed to sevoflurane and opioid in the SGA group. This means that we cannot determine whether the positive effect was related to the epidural, the use of propofol, the reduction in opioid use, or all of these. In a future study, we will compare propofol-based total intravenous with volatile anaesthesia, and, depending on the outcome of that, concomitant use of epidural or opioid analgesia.

In conclusion, our results suggest that anaesthetic technique might affect the serum milieu and therefore the potential for tumour metastasis in patients with colon cancer undergoing surgery.

Acknowledgements

No competing interests declared.